Aptamers for the treatment of sickle cell disease

ABSTRACT

The present invention provides polynucleotide aptamers that selectively bind to and inhibit polymerization of sickle hemoglobin (HbS), pharmaceutical compositions comprising the same, methods of use for diagnostics and treatment of sickle cell disease, methods of use as capture reagents, and methods of rational drug design.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/828,142, filed May 28, 2013; which is incorporated herein byreference in its entirety.

BACKGROUND

Sickle cell anemia (SCA), a genetic disorder affecting 1 in 400 AfricanAmericans and up to 2% of the population in some areas of Africa,results from the production of an abnormal type of hemoglobin thatpolymerizes (aggregates) leading to detrimental shape changes in redblood cells (sickling) and significant morbidity and mortality inpatients.

Polymerization of sickle hemoglobin (HbS) in the red blood cells ofpatients with SCA leads to rigid red cells which occlude blood vessels,leading to pain, strokes, organ damage, susceptibility to infection andearly death. Present methods known in the art that have been shown toalter the severity of the disorder are complex and labor intensivetherapies: 1) bone marrow transplantation; 2) routine bloodtransfusions; or 3) hydroxyurea, a drug which indirectly (andincompletely) prevents HbS polymerization by inducing the production ofanother type of hemoglobin (fetal hemoglobin). Accordingly, treatmentsfor SCA are lacking and focus mainly on palliative or symptomatictherapy.

SUMMARY

In some aspects, the presently disclosed subject matter providespolynucleotide aptamers that specifically bind sickle hemoglobin (HbS)in such a way that polymerization of HbS is inhibited without adeleterious effect on hemoglobin's functional capabilities. In certainaspects, the polynucleotide aptamers are RNA aptamers. In other aspects,polynucleotide aptamers inhibit polymerization of HbS. Thepolynucleotide aptamers may specifically bind oxygenated HbS (oxy-HbS),deoxygenated HbS (deoxy-HbS), or may bind both oxy-HbS and deoxy-HbS. Incertain aspects, the polynucleotide aptamers comprise a nucleotidesequence that is at least 70% identical, e.g., at least 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any oneof SEQ ID NOS:2-60, particularly SEQ ID NOS:2, 4, 31, and 37 orfragments or analogs thereof, more particularly: (a) any one of SEQ IDNOS:2, 4, or 5 wherein the polynucleotide aptamer further comprises aconsensus sequence consisting of SEQ ID NO:61; (b) any one of SEQ IDNOS:11 or 14 or wherein the polynucleotide aptamer further comprises aconsensus sequence consisting of SEQ ID NO:62; (c) any one of SEQ IDNOS:37, 38, 40, or 49 wherein the polynucleotide further comprises aconsensus sequence consisting of SEQ ID NO:63; (d) any one of SEQ IDNOS:31, 37, 38, 40, 42, 45, 46, 47, 48, 49, 53, 56, 59, or 60 whereinthe polynucleotide aptamer further comprises a consensus sequenceconsisting of SEQ ID NO:64; or (e) any one of SEQ ID NOS:2, 4, 5, 8, 34,or 57 wherein the polynucleotide aptamer further comprises a consensussequence consisting of SEQ ID NO:65. In another aspect, thepolynucleotide aptamer of the presently disclosed subject mattercomprises a consensus sequence consisting of a nucleotide sequenceselected from the group consisting of SEQ ID NOS:61, 62, 63, 64, and 65.Other aspects of the presently disclosed subject matter relate topolynucleotides encoding the polynucleotide aptamers of the invention,vectors comprising the polynucleotide aptamers, and cells comprising thepolynucleotide aptamers.

In other aspects, the presently disclosed subject matter provides amethod of treating or preventing sickle cell disease in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of a polynucleotide aptamer thatspecifically binds sickle hemoglobin (HbS), where the polynucleotideaptamer inhibits polymerization of HbS. In certain aspects, thepolynucleotide aptamers are modified to increase the circulatinghalf-life of the aptamer after administration to a subject. In anotheraspect, the polynucleotide aptamer is administered in a pharmaceuticallyacceptable carrier. In other aspects, the sickle cell disease is sicklecell anemia. In yet another aspect, the method of treating or preventingsickle cell disease further comprises contacting the polynucleotideaptamer with an antidote, particularly an oligonucleotide comprising asequence complementary to at least a portion of the polynucleotideaptamer.

In other aspects, the presently disclosed subject matter provides amethod for diagnosing or predicting sickle cell disease in a subjecthaving or at risk of developing sickle cell disease or at risk ofpassing it on to offspring, the method comprising: (a) obtaining abiological sample from the subject; (b) contacting the biological samplewith a polynucleotide aptamer that specifically binds to HbS; and (c)detecting binding of the polynucleotide aptamer with HbS in thebiological sample; where detection of binding of the polynucleotideaptamer with HbS in the biological sample is indicative of the subjecthaving or at risk of developing sickle cell disease or at risk ofpassing it on to offspring. In one aspect, the sickle cell disease issickle cell anemia. In another aspect, the biological sample compriseswhole blood, hemocytes, serum, or plasma. In other aspects, thepolynucleotide aptamer is labeled for detection with a fluorescent,luminescent, phosphorescent, radioactive, or colorimetric compound.

In yet other aspects, the presently disclosed subject matter provides amethod of purifying hemoglobin from a biological sample, the methodcomprising: (a) providing a biological sample containing hemoglobin; (b)contacting the biological sample with a polynucleotide aptamer thatspecifically binds to HbS under conditions effective to bind hemoglobinto the aptamer; and (c) recovering the hemoglobin bound to the aptamer.In one aspect, the step of contacting the biological sample with thepolynucleotide aptamer that specifically binds to HbS comprisesproviding a solid support comprising an aptamer that specifically bindsto HbS immobilized onto the solid support through a spacer. In otheraspects, the polynucleotide aptamer is modified to enable covalentimmobilization or to prevent enzymatic degradation. In another aspect,the biological sample comprises whole blood, hemocytes, serum, orplasma.

In other aspects, the presently disclosed subject matter provides amethod of using a three-dimensional structure of a polynucleotideaptamer that specifically binds to HbS and inhibits polymerization ofHbS in a drug screening assay comprising: (a) selecting a potential drugby performing rational drug design with the three-dimensional structureof the polynucleotide aptamer that specifically binds to HbS andinhibits polymerization of HbS determined from one or more sets ofatomic coordinates; wherein said selecting is performed in conjunctionwith computer modeling; (b) contacting the potential drug with HbS; (c)detecting the binding of the potential drug with the HbS; and (d)detecting the inhibition of polymerization of HbS by the potential drug;wherein a potential drug is selected as a drug if the potential drugbinds to HbS and inhibits polymerization of HbS.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIG. 1 shows dose-dependent saturable binding of deoxy aptamer to deoxysickle hemoglobin (HbS);

FIG. 2 shows inhibition of HbS polymerization by deoxy 3-A aptamer (SEQID NO:4) in a polymerization assay;

FIG. 3 shows inhibition of HbS polymerization by deoxy 1 aptamer (SEQ IDNO:2) in one of two runs of a polymerization assay;

FIG. 4 shows inhibition of HbS polymerization by deoxy EM8-A aptamer(SEQ ID NO:31) in a polymerization assay;

FIG. 5 shows inhibition of HbS polymerization by oxy 3-B aptamer (SEQ IDNO:37) in a polymerization assay;

FIGS. 6A-6B show the concentration-dependent inhibition of HbSpolymerization by deoxy 3-A aptamer: A) polymerization curves as afunction of deoxy 3-A aptamer concentration; and B) slope ofpolymerization curves;

FIG. 7 shows that lipofectin facilitates entry of deoxy 3-A aptamer intosickle red blood cells; and

FIG. 8 shows that HbS retains the ability to form new polymer whengrowing filament ends are provided by mechanical disruption.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the presently disclosed subject matter areshown. Like numbers refer to like elements throughout. The presentlydisclosed subject matter may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements. Indeed, many modifications andother embodiments of the presently disclosed subject matter set forthherein will come to mind to one skilled in the art to which thepresently disclosed subject matter pertains having the benefit of theteachings presented in the foregoing descriptions and the associatedFigures. Therefore, it is to be understood that the presently disclosedsubject matter is not to be limited to the specific embodimentsdisclosed and that modifications and other embodiments are intended tobe included within the scope of the appended claims.

I. Hemoglobins and Sickle Cell Disease

Normally, hemoglobin is a tetrameric protein composed of two pairs oftwo different subunits. Hemoglobin A (hereinafter abbreviated as HbA)has α-chain and β-chain subunits. Binding of glucose to N-terminal aminoacid(s) of this/these β-chain results in hemoglobin A_(1c) (hereinafterabbreviated as HbA_(1c)). HbA_(1c), if produced via a reversiblereaction therebetween, is called labile HbA_(1c) and, if produced via anirreversible reaction involving the labile HbA_(1c), is called stableHbA_(1c).

The separation of hemoglobins present in a hemolyzed sample by means ofcation exchange liquid chromatography, if performed over a sufficientlylong period of time, generally results in the sequential elution ofhemoglobin A_(1a) (hereinafter abbreviated as HbA_(1a)) and hemoglobinA_(1b) (hereinafter abbreviated as HbA_(1b)), hemoglobin F (hereinafterabbreviated as HbF), labile HbA_(1c), stable HbA_(1c) and hemoglobin A₀(hereinafter abbreviated as HbA₀). HbA_(1a), HbA_(1b) and HbA_(1c) eachis a glycated HbA. HbF is fetal hemoglobin composed of α and γ chains.HbA₀ consists of a group of hemoglobin components, includes HbA as itsprimary component and is retained more strongly to a column thanHbA_(1c).

Hemoglobin S or sickle hemoglobin (hereinafter abbreviated as HbS) andhemoglobin C (hereinafter abbreviated as HbC) are known as “abnormalhemoglobins.” HbS and HbC result from substitution of glutamic acidlocated in a sixth position from an N-terminal of the β chain of HbA forvaline and lysine, respectively. Hemoglobin A₂ (hereinafter abbreviatedas HbA₂) is composed of a and δ chains and, like HbF, its elevated levelis interpreted as evidence of Mediterranean anemia (thalassemia). In thenormal determination of hemoglobins by cation exchange liquidchromatography, they are eluted in the sequence of HbA₀, HbA₂, HbS andHbC.

Sickle-cell disease (SCD), or sickle-cell anemia (or drepanocytosis), isa life-long blood disorder characterized by red blood cells(erythrocytes: RBC) that assume an abnormal, rigid, sickle shape.Sickling decreases flexibility of RBC and results in a risk of variouscomplications. RBC sickling occurs because of a mutation in thehemoglobin gene. SCD is an inherited disorder and SCD is an autosomalrecessive disease. Although, some people who inherit one sickle cellgene and one other defective hemoglobin gene may experience a similarsickle-cell disorder. Sickle cell disease includes but is not limited tosickle cell anemia, sickle β-thalassemia, sickle cell-hemoglobin Cdisease and any other sickle hemoglobinopathy in which HbS interactswith a hemoglobin other than HbS. “Sickle hemoglobinopathy” is anabnormality of hemoglobin which results in sickle cell disease or sicklevariants.

II. Polynucleotide Aptamers

A. Aptamers

In some embodiments, the presently disclosed subject matter relates tothe generation of aptamers that specifically bind sickle hemoglobin(HbS) in such a way that polymerization of HbS is inhibited without adeleterious effect on hemoglobin's functional capabilities. Aptamers aresmall single-stranded nucleic acid molecules (˜5-25 kDa) that fold intounique structures, allowing them to bind to molecular targets with highspecificity and affinity. This specific binding confers the potentialfor aptamers to be used in a wide variety of diagnostic or therapeuticapplications and have emerged as viable alternatives to small-moleculeand antibody-based therapy (Que-Gewirth et al. (2007) Gene Ther. 14:283;Ireson et al. (2006) Mol. Cancer Ther. 5:2957). Like antibodies,aptamers possess binding affinities in the low nanomolar to picomolarrange. However, aptamers are advantageous in that they are easilysynthesized and stored, can bind very small targets, arenon-immunogenic, are heat stable, possess minimal interbatchvariability, and can be antidote-controlled. In addition, in contrast toantisense oligonucleotides, RNA aptamers can effectively targetextracellular targets, such as HbS. Furthermore, chemical modifications,such as amino or fluoro substitutions at the 2′ position of pyrimidines,may reduce degradation by nucleases. The biodistribution and clearanceof aptamers can also be altered by chemical addition of moieties such aspolyethylene glycol and cholesterol.

An aptamer's small size also maximizes its ability to bind to a specificsite on a protein, altering the function of that site, without affectingthe functions of other sites on the protein. For example, Fortenberryand colleagues have developed aptamers that bind specifically toplasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitorthat has a role in the pathophysiology of several diseases, includingcancer and cardiovascular disease. PAI-1 binds to vitronectin,preventing vitronectin's interaction with integrin, thereby resulting ina decrease in cell adhesion and migration. These aptamers bindspecifically to PAI-1's vitronectin binding site, affecting PAI-1'sinteraction with vitronectin, but having no affect on its proteolyticactivity.

Specific aptamers are typically selected from very large libraries ofmore than 10¹⁴ random sequence oligonucleotides in a process called the“systematic evolution of ligands by exponential enrichment” (SELEX).This is an iterative selection process, which begins with a protein orother target of interest being incubated with the oligonucleotidelibrary. A small fraction of the oligonucleotides bind the target andthe rest are separated out by a suitable separation technique. The smallpopulation that bound the target is then amplified and used in the nextround of incubation with the target. This cycle is repeated multipletimes, with increasingly stringent incubation and separation conditionsat each round in order to enrich for high affinity binders. This processwill be referred to herein as “positive selection.” However, in certaincases the aptamers that do not bind to the target protein will beamplified, and the binders will be discarded. This process will bereferred to herein as “negative selection.” Both positive and negativeselection may be used, including experiments where the bound aptamersare recovered from the target—positive selection, and experiments wherethe aptamers that recognized more than one target are removed from thepool (for example, by absorbing pools of molecules that bind both HbAand HbS to HbA, allowing the molecules that specifically bind only toHbS to “flow through”—negative selection).

Accordingly, in one embodiment, the presently disclosed subject matterrelates to polynucleotide aptamers that specifically bind to sicklehemoglobin (HbS). In some embodiments, the polynucleotide aptamersinhibit the polymerization of HbS, particularly without a deleteriouseffect on hemoglobin's functional capabilities. Preventing thepolymerization of HbS is essentially a cure for sickle cell anemia,since the complications of the disease arise directly as a result of redblood cell changes brought about by HbS polymerization.

As used herein, “polymerization” includes the process of forming apolymer from many monomeric units of hemoglobin. A polymer may be formedby any chemical bonding interaction between or among molecules, i.e.covalent, ionic, or van der Waals. As used herein, “aggregation” and“polymerization” may be used interchangeably. In particular embodiments,the presently disclosed aptamers inhibit polymerization of HbS. However,it is understood by those of skill in the art that 100% inhibition ofpolymerization of HbS is not required within the presently disclosedmethods. In some embodiments, the presently disclosed methods produce atleast about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% inhibition ofpolymerization of HbS relative to polymerization of HbS measured inabsence of aptamers or modified aptamers described herein thatspecifically bind HbS and inhibit polymerization of HbS, i.e., a controlsample, in an assay.

Thus, one embodiment of the presently disclosed subject matter relatesto polynucleotide aptamers that specifically bind to HbS and inhibitpolymerization of HbS, particularly without a deleterious effect onhemoglobin's functional capabilities. In another embodiment, thepresently disclosed subject matter relates to polynucleotide aptamersthat specifically bind to oxygenated HbS (oxy-HbS) or to deoxygenatedHbS (deoxy-HbS), or that specifically bind to both oxy-HbS anddeoxy-HbS. In one embodiment, the aptamers are DNA or RNA aptamers orhybrid DNA/RNA aptamers. In a particular embodiment, the aptamers areRNA aptamers.

The term “specifically binds,” as used herein, refers to a molecule(e.g., an aptamer) that binds to a target (e.g., a protein such as HbS)with at least five-fold greater affinity as compared to any non-targets,e.g., at least 10-, 20-, 50-, or 100-fold greater affinity. The aptamersof the presently disclosed subject matter may bind HbS, includingoxy-HbS and/or deoxy-HbS, as well as HbA and other types of hemoglobin,with a K_(d) of less than about 1000 nM, e.g., less than about 500, 200,100, 50, or 20 nM.

The sequence of the polynucleotide aptamers of the invention may beselected by any method known in the art. In one embodiment, aptamers areselected by the SELEX process. In another embodiment, aptamers may beselected by starting with the sequences and structural requirements ofthe aptamers disclosed herein and modifying the sequences to produceother aptamers.

The length of the aptamers of the presently disclosed subject matter isnot limited, but typical aptamers have a length of about 10 to about 120nucleotides, particularly about 80 nucleotides. In certain embodiments,the aptamer may have additional nucleotides attached to the 5′- and/or3′ end. The additional nucleotides may be, e.g., part of primersequences, restriction endonuclease sequences, or vector sequencesuseful for producing the aptamer.

The polynucleotide aptamers of the present invention may be comprised ofribonucleotides only (RNA aptamers), deoxyribonucleotides only (DNAaptamers), or a combination of ribonucleotides and deoxyribonucleotides.The nucleotides may be naturally occurring nucleotides (e.g., ATP, TTP,GTP, CTP, UTP) or modified nucleotides. Modified nucleotides refers tonucleotides comprising bases such as, for example, adenine, guanine,cytosine, thymine, and uracil, xanthine, inosine, and queuosine thathave been modified by the replacement or addition of one or more atomsor groups. Some examples of types of modifications that can comprisenucleotides that are modified with respect to the base moieties, includebut are not limited to, alkylated, halogenated, thiolated, aminated,amidated, or acetylated bases, in various combinations. More specificexamples include 5-propynyluridine, 5-propynylcytidine, 6-methyladenine,6-methylguanine, N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine,2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine,5-methyluridine and other nucleotides having a modification at the 5position, 5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine,4-acetylcytidine, 1-methyladenosine, 2-methyladenosine,3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine,2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine,deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthylgroups, any 0- and N-alkylated purines and pyrimidines such asN6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyaceticacid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groupssuch as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines thatact as G-clamp nucleotides, 8-substituted adenines and guanines,5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkylnucleotides, carboxyalkylaminoalkyl nucleotides, andalkylcarbonylalkylated nucleotides. Modified nucleotides also includethose nucleotides that are modified with respect to the sugar moiety(e.g., 2′-fluoro or 2′-O-methyl nucleotides), as well as nucleotideshaving sugars or analogs thereof that are not ribosyl. For example, thesugar moieties may be, or be based on, mannoses, arabinoses,glucopyranoses, galactopyranoses, 4′-thioribose, and other sugars,heterocycles, or carbocycles. The term nucleotide is also meant toinclude what are known in the art as universal bases. By way of example,universal bases include but are not limited to 3-nitropyrrole,5-nitroindole, or nebularine. Modified nucleotides include labelednucleotides such as radioactively, enzymatically, or chromogenicallylabeled nucleotides).

In one embodiment, the presently disclosed subject matter relates to anRNA aptamer and comprises a nucleotide sequence that is identical to anyone of SEQ ID NOS:2-60 as shown in Table 1 (see Example 1). In anotherembodiment, the RNA aptamer consists of a nucleotide sequence that isidentical to any one of SEQ ID NOS:2-60. In a further embodiment, theRNA aptamer comprises a nucleotide sequence that is at least 70%identical, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS:2-60. Inanother embodiment, the aptamer consists of a nucleotide sequence thatis at least 70% identical, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ IDNOS:2-60. In yet another embodiment, the aptamer comprises a nucleotidesequence that is identical to a fragment of any one of SEQ ID NOS:2-60of at least 10 contiguous nucleotides, e.g., at least about 15, 20, 25,30, or 35 contiguous nucleotides. In another embodiment, the aptamercomprises a nucleotide sequence that is at least 70% identical, e.g., atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%; 96%, 97%, 98%, or 99%identical to a fragment of any one of SEQ ID NOS: 2-60 of at leastcontiguous 10 nucleotides, e.g., at least about 15, 20, 25, 30, or 35contiguous nucleotides. In one embodiment, one or more ribonucleotidesin the RNA aptamers described above are substituted by adeoxyribonucleotide. In another embodiment, the fragments and/or analogsof the aptamers of SEQ ID NOS:2-60 have a substantially similar activityas one or more of the aptamers of SEQ ID NOS:2-60. “Substantiallysimilar,” as used herein, refers to specific binding to HbS, and in someembodiments also refers to an inhibitory activity on the polymerizationof HbS, particularly without a deleterious effect on hemoglobin'sfunctional capabilities, that is at least about 20% of the inhibitoryactivity of one or more of the aptamers of SEQ ID NOS:2-60.

Changes to the aptamer sequences, such as SEQ ID NOS:2-60, may be madebased on structural requirements for binding of the aptamers to HbS,including oxy-HbS and/or deoxy-HbS. The structural requirements may bereadily determined by one of skill in the art by analyzing commonsequences between the disclosed aptamers and/or by mutagenizing thedisclosed aptamers and measuring HbS binding affinity.

When a number of individual, distinct aptamer sequences for a singletarget molecule have been obtained and sequenced as described herein,the sequences can be examined for “consensus sequences.” As used herein,“consensus sequence” refers to a nucleotide sequence or region (whichmight or might not be made up of contiguous nucleotides) that is foundin one or more regions of at least two aptamers, the presence of whichcan be correlated with aptamer-to-target-binding or with aptamerstructure.

A consensus sequence can be as short as three nucleotides long. It alsocan be made up of one or more noncontiguous sequences with nucleotidesequences or polymers of hundreds of bases long interspersed between theconsensus sequences. Consensus sequences can be identified by sequencecomparisons between individual aptamer species, which comparisons can beaided by computer programs and other tools for modeling secondary andtertiary structure from sequence information. Generally, the consensussequence will contain at least about 5 to 20 nucleotides, more commonlyfrom 11 to 15 nucleotides.

In one embodiment, wherein when the RNA aptamer of the presentlydisclosed subject matter comprises a nucleotide sequence that is atleast 70% identical, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS:2, 4,or 5 or fragments or analogs thereof, the RNA aptamer further comprisesa consensus sequence consisting of GAACUGGGCUG (SEQ ID NO:61).

In another embodiment, wherein when the RNA aptamer of the presentlydisclosed subject matter comprises a nucleotide sequence that is atleast 70% identical, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS:11 or14 or fragments or analogs thereof, the RNA aptamer further comprises aconsensus sequence consisting of CACCCCAACGCGGAG (SEQ ID NO:62).

In another embodiment, wherein when the RNA aptamer of the presentlydisclosed subject matter comprises a nucleotide sequence that is atleast 70% identical, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS:37,38, 40, or 49, or fragments or analogs thereof, the RNA aptamer furthercomprises a consensus sequence consisting of GUCUAUUAGGAC (SEQ IDNO:63).

In another embodiment, wherein when the RNA aptamer of the presentlydisclosed subject matter comprises a nucleotide sequence that is atleast 70% identical, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS:31,37, 38, 40, 42, 45, 46, 47, 48, 49, 53, 56, 59, or 60 or fragments oranalogs thereof, the RNA aptamer further comprises a consensus sequenceconsisting of CUAUUAGGACCAG (SEQ ID NO:64).

In another embodiment, wherein when the RNA aptamer of the presentlydisclosed subject matter comprises a nucleotide sequence that is atleast 70% identical, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS:2, 4,5, 8, 34, or 57, or fragments or analogs thereof, the RNA aptamerfurther comprises a consensus sequence consisting of CGAUUAGAACUGG (SEQID NO:65).

In another embodiment, the RNA aptamer of the presently disclosedsubject matter comprises a consensus sequence consisting of a nucleotidesequence selected from the group consisting of SEQ ID NOS:61, 62, 63,64, and 65.

As used herein, a “nucleic acid” or “polynucleotide” refers to thephosphate ester polymeric form of ribonucleosides (adenosine, guanosine,uridine or cytidine; “RNA molecules”) or deoxyribonucleosides(deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNAmolecules”), or any phosphoester anologs thereof, such asphosphorothioates and thioesters, in either single stranded form, or adouble-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNAhelices are possible. The term nucleic acid molecule, and in particularDNA or RNA molecule, refers only to the primary and secondary structureof the molecule, and does not limit it to any particular tertiary forms.Thus, this term includes double-stranded DNA found, inter alia, inlinear or circular DNA molecules (e.g., restriction fragments),plasmids, and chromosomes. In discussing the structure of particulardouble-stranded DNA molecules, sequences may be described hereinaccording to the normal convention of giving only the sequence in the 5′to 3′ direction along the non-transcribed strand of DNA (i.e., thestrand having a sequence homologous to the mRNA). A “recombinant DNAmolecule” is a DNA molecule that has undergone a molecular biologicalmanipulation.

The term “fragment” refers to a nucleotide sequence of reduced lengthrelative to the reference nucleic acid and comprising, over the commonportion, a nucleotide sequence identical to the reference nucleic acid.Such a nucleic acid fragment according to the presently disclosedsubject matter may be, where appropriate, included in a largerpolynucleotide of which it is a constituent. Such fragments comprise, oralternatively consist of, oligonucleotides ranging in length from atleast 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42,45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120,135, 150, 200, 300, 500, 720, 900, 1000 or 1500 consecutive nucleotidesof a nucleic acid according to the presently disclosed subject matter.

The term “percent identity,” as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesmay be performed using the Clustal method of alignment (Higgins andSharp (1989) CABIOS. 5:151-153) with the default parameters, includingdefault parameters for pairwise alignments.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include but is not limited to the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.),BLASTP, BLASTN, BLASTX (Altschul et al. (1990) J. Mol. Biol.215:403-410, and DNASTAR (DNASTAR, Inc., Madison, Wis.). Within thecontext of this application it will be understood that where sequenceanalysis software is used for analysis, that the results of the analysiswill be based on the “default values” of the program referenced, unlessotherwise specified. As used herein “default values” will mean any setof values or parameters which originally load with the software whenfirst initialized.

The term “isolated” designates a biological material (nucleic acid orprotein) that has been removed from its original environment (theenvironment in which it is naturally present). For example, apolynucleotide present in the natural state in a plant or an animal isnot isolated, however the same polynucleotide separated from theadjacent nucleic acids in which it is naturally present, is considered“isolated”. The term “purified” does not require the material to bepresent in a form exhibiting absolute purity, exclusive of the presenceof other compounds.

B. Polynucleotides Encoding Aptamers, Vectors, and Cells

Once an aptamer sequence according to the presently disclosed subjectmatter is identified, the aptamer may by synthesized by any method knownto those of skill in the art. In one embodiment, aptamers may beproduced by chemical synthesis of oligonucleotides and/or ligation ofshorter oligonucleotides. Accordingly, another embodiment of the presentinvention relates to polynucleotides encoding the aptamers of theinvention. The polynucleotides may be used to express the aptamers,e.g., by in vitro transcription, polymerase chain reactionamplification, or cellular expression. The polynucleotide may be DNAand/or RNA and may be single-stranded or double-stranded. In oneembodiment, the polynucleotide is a vector which may be used to expressthe aptamer. The vector may be, e.g., a plasmid vector or a viral vectorand may be suited for use in any type of cell, such as mammalian,insect, plant, fungal, or bacterial cells. The vector may comprise oneor more regulatory elements necessary for expressing the aptamers, e.g.,a promoter, enhancer, transcription control elements, etc. Anotherembodiment of the invention relates to a cell comprising apolynucleotide encoding the aptamers of the invention. In anotherembodiment, the invention relates to a cell comprising the aptamers ofthe invention. The cell may be any type of cell, e.g., mammalian,insect, plant, fungal, or bacterial cells.

Several methods known in the art may be used to propagate apolynucleotide according to the presently disclosed subject matter. Oncea suitable host system and growth conditions are established,recombinant expression vectors can be propagated and prepared inquantity. As described herein, the expression vectors which can be usedinclude, but are not limited to, the following vectors or theirderivatives: human or animal viruses such as vaccinia virus oradenovirus; insect viruses such as baculovirus; yeast vectors;bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNAvectors, to name but a few.

A “vector” is any means for the cloning of and/or transfer of a nucleicacid into a host cell. A vector may be a replicon to which another DNAsegment may be attached so as to bring about the replication of theattached segment. A “replicon” is any genetic element (e.g., plasmid,phage, cosmid, chromosome, virus) that functions as an autonomous unitof DNA replication in vivo, i.e., capable of replication under its owncontrol. The term “vector” includes both viral and nonviral means forintroducing the nucleic acid into a cell in vitro, ex vivo or in vivo. Alarge number of vectors known in the art may be used to manipulatenucleic acids, incorporate response elements and promoters into genes,etc. Possible vectors include, for example, plasmids or modified virusesincluding, for example bacteriophages such as lambda derivatives, orplasmids such as pBR322 or pUC plasmid derivatives, or the Bluescriptvector. For example, the insertion of the DNA fragments corresponding toresponse elements and promoters into a suitable vector can beaccomplished by ligating the appropriate DNA fragments into a chosenvector that has complementary cohesive termini. Alternatively, the endsof the DNA molecules may be enzymatically modified or any site may beproduced by ligating nucleotide sequences (linkers) into the DNA terminiSuch vectors may be engineered to contain selectable marker genes thatprovide for the selection of cells that have incorporated the markerinto the cellular genome. Such markers allow identification and/orselection of host cells that incorporate and express the proteinsencoded by the marker.

Viral vectors, and particularly retroviral vectors, have been used in awide variety of gene delivery applications in cells, as well as livinganimal subjects. Viral vectors that can be used include but are notlimited to retrovirus, adeno-associated virus, pox, baculovirus,vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, andcaulimovirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers. In addition to a nucleic acid, a vector may also compriseone or more regulatory regions, and/or selectable markers useful inselecting, measuring, and monitoring nucleic acid transfer results(transfer to which tissues, duration of expression, etc.).

Vectors may be introduced into the desired host cells by methods knownin the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al. (1992) J. Biol. Chem.267:963; Wu et al. (1988) J. Biol. Chem. 263:14621). Aptamers may alsobe targeted to cells of interest by coupling aptamers to other aptamersthat are known to specifically enter cells of interest, which can bescreened for, or by attachment to other ligands for red cell receptorsthat are internalized (e.g., transferrin-transferrin receptors), asdescribed more fully below.

A polynucleotide according to the presently disclosed subject matter canalso be introduced in vivo by lipofection. For the past decade, therehas been increasing use of liposomes for encapsulation and transfectionof nucleic acids in vitro. Synthetic cationic lipids designed to limitthe difficulties and dangers encountered with liposome-mediatedtransfection can be used to prepare liposomes for in vivo transfectionof a gene encoding a marker (Feigner et al. (1988) Proc. Natl. Acad.Sci. USA 84:7413; Mackey et al. (1988) Proc. Natl. Acad. Sci. U.S.A.85:8027; and Ulmer et al. (1993) Science 259:1745). The use of cationiclipids may promote encapsulation of negatively charged nucleic acids,and also promote fusion with negatively charged cell membranes (Feigneret al. (1989) Science 337:387). Particularly useful lipid compounds andcompositions for transfer of nucleic acids are described in PCT PatentPubs. WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127. The useof lipofection to introduce exogenous genes into the specific organs invivo has certain practical advantages. Molecular targeting of liposomesto specific cells represents one area of benefit. It is clear thatdirecting transfection to particular cell types would be particularlypreferred in a tissue with cellular heterogeneity, such as pancreas,liver, kidney, and the brain. Lipids may be chemically coupled to othermolecules for the purpose of targeting (Mackey et al. (1988) Proc. Natl.Acad. Sci. U.S.A. 85:8027). Targeted peptides, e.g., hormones orneurotransmitters, and proteins such as antibodies, or non-peptidemolecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g., PCT PatentPub. WO95/21931), peptides derived from DNA binding proteins (e.g., PCTPatent Pub. WO96/25508), or a cationic polymer (e.g., PCT Patent Pub.WO95/21931).

It is also possible to introduce a vector in vivo as a naked DNA plasmid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated DNA delivery approaches can also be used (Curiel etal. (1992) Hum. Gene Ther. 3:147; Wu et al. (1987) J. Biol. Chem.262:4429).

The term “transfection” means the uptake of exogenous or heterologousRNA or DNA by a cell. A cell has been “transfected” by exogenous orheterologous RNA or DNA when such RNA or DNA has been introduced insidethe cell. A cell has been “transformed” by exogenous or heterologous RNAor DNA when the transfected RNA or DNA effects a phenotypic change. Thetransforming RNA or DNA can be integrated (covalently linked) intochromosomal DNA making up the genome of the cell.

The term “promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters.” Promotersthat cause a gene to be expressed in a specific cell type are commonlyreferred to as “cell-specific promoters” or “tissue-specific promoters.”Promoters that cause a gene to be expressed at a specific stage ofdevelopment or cell differentiation are commonly referred to as“developmentally-specific promoters” or “cell differentiation-specificpromoters.” Promoters that are induced and cause a gene to be expressedfollowing exposure or treatment of the cell with an agent, biologicalmolecule, chemical, ligand, light, or the like that induces the promoterare commonly referred to as “inducible promoters” or “regulatablepromoters.” It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of different lengths may have identical promoter activity.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentlydisclosed subject matter, the promoter sequence is bounded at its 3′terminus by the transcription initiation site and extends upstream (5′direction) to include the minimum number of bases or elements necessaryto initiate transcription at levels detectable above background. Withinthe promoter sequence will be found a transcription initiation site(conveniently defined for example, by mapping with nuclease S1), as wellas protein binding domains (consensus sequences) responsible for thebinding of RNA polymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced (if the coding sequence contains introns) and translated intothe protein encoded by the coding sequence.

“Transcriptional and translational control sequences” are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

Enhancers that may be used in embodiments of the presently disclosedsubject matter include but are not limited to: an SV40 enhancer, acytomegalovirus (CMV) enhancer, an elongation factor I (EF1) enhancer,yeast enhancers, viral gene enhancers, and the like.

Termination control regions, i.e., terminator or polyadenylationsequences, may also be derived from various genes native to thepreferred hosts. In one embodiment of the presently disclosed subjectmatter, the termination control region may comprise or be derived from asynthetic sequence, synthetic polyadenylation signal, an SV40 latepolyadenylation signal, an SV40 polyadenylation signal, a bovine growthhormone (BGH) polyadenylation signal, viral terminator sequences, or thelike.

The terms “3′ non-coding sequences” or “3′ untranslated region (UTR)”refer to DNA sequences located downstream (3′) of a coding sequence andmay comprise polyadenylation [poly(A)] recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor.

The term “regulatory region” means a nucleic acid sequence thatregulates the expression of a second nucleic acid sequence. A regulatoryregion may include sequences which are naturally responsible forexpressing a particular nucleic acid (a homologous region) or mayinclude sequences of a different origin that are responsible forexpressing different proteins or even synthetic proteins (a heterologousregion). In particular, the sequences can be sequences of prokaryotic,eukaryotic, or viral genes or derived sequences that stimulate orrepress transcription of a gene in a specific or non-specific manner andin an inducible or non-inducible manner. Regulatory regions includeorigins of replication, RNA splice sites, promoters, enhancers,transcriptional termination sequences, and signal sequences which directthe polypeptide into the secretory pathways of the target cell.

C. Modified Aptamers

In one embodiment of the presently disclosed subject matter, theaptamers are modified to increase the circulating half-life of theaptamer after administration to a subject. In one embodiment, thenucleotides of the aptamers are linked by phosphate linkages. In anotherembodiment, one or more of the internucleotide linkages are modifiedlinkages, e.g., linkages that are resistant to nuclease degradation. Theterm “modified internucleotide linkage” includes all modifiedinternucleotide linkages known in the art or that come to be known andthat, from reading this disclosure, one skilled in the art will concludeis useful in connection with the present invention. Internucleotidelinkages may have associated counterions, and the term is meant toinclude such counterions and any coordination complexes that can form atthe internucleotide linkages. Modifications of internucleotide linkagesinclude, without limitation, phosphorothioates, phosphorodithioates,methylphosphonates, 5′-alkylenephosphonates, 5′-methylphosphonate,3′-alkylene phosphonates, borontrifluoridates, borano phosphate estersand selenophosphates of 3′-5′ linkage or 2′-5′ linkage,phosphotriesters, thionoalkylphosphotriesters, hydrogen phosphonatelinkages, alkyl phosphonates, alkylphosphonothioates,arylphosphonothioates, phosphoroselenoates, phosphorodiselenoates,phosphinates, phosphoramidates, 3′-alkylphosphoramidates,aminoalkylphosphoramidates, thionophosphoramidates,phosphoropiperazidates, phosphoroanilothioates, phosphoroanilidates,ketones, sulfones, sulfonamides, carbonates, carbamates,methylenehydrazos, methylenedimethylhydrazos, formacetals,thioformacetals, oximes, methyleneiminos, methylenemethyliminos,thioamidates, linkages with riboacetyl groups, aminoethyl glycine, silylor siloxane linkages, alkyl or cycloalkyl linkages with or withoutheteroatoms of, for example, 1 to 10 carbons that can be saturated orunsaturated and/or substituted and/or contain heteroatoms, linkages withmorpholino structures, amides, polyamides wherein the bases can beattached to the aza nitrogens of the backbone directly or indirectly,and combinations of such modified internucleotide linkages. In anotherembodiment, the aptamers comprise 5′- or 3′-terminal blocking groups toprevent nuclease degradation (e.g., an inverted deoxythymidine orhexylamine).

In a further embodiment, the aptamers are linked to conjugates thatincrease the circulating half-life, e.g., by decreasing nucleasedegradation or renal filtration of the aptamer. Conjugates may include,for example, amino acids, peptides, polypeptides, proteins, antibodies,antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars,carbohydrates, polymers such as polyethylene glycol and polypropyleneglycol, as well as analogs or derivatives of all of these classes ofsubstances. Additional examples of conjugates also include steroids,such as cholesterol, phospholipids, di- and tri-acylglycerols, fattyacids, hydrocarbons that may or may not contain unsaturation orsubstitutions, enzyme substrates, biotin, digoxigenin, andpolysaccharides. Further examples include thioethers such ashexyl-S-tritylthiol, thiocholesterol, acyl chains such as dodecandiol orundecyl groups, phospholipids such as di-hexadecyl-rac-glycerol,triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate,polyamines, polyethylene glycol, adamantane acetic acid, palmitylmoieties, octadecylamine moieties, hexylaminocarbonyl-oxycholesterol,farnesyl, geranyl and geranylgeranyl moieties, such as polyethyleneglycol, cholesterol, lipids, or fatty acids. Conjugates can also bedetectable labels. For example, conjugates can be fluorophores.Conjugates can include fluorophores such as TAMRA, BODIPY, cyaninederivatives such as Cy3 or Cy5 Dabsyl, or any other suitable fluorophoreknown in the art. A conjugate may be attached to any position on theterminal nucleotide that is convenient and that does not substantiallyinterfere with the desired activity of the aptamer that bears it, forexample the 3′ or 5′ position of a ribosyl sugar. A conjugatesubstantially interferes with the desired activity of an aptamer if itadversely affects its functionality such that the ability of the aptamerto bind HbS, including oxy-HbS and/or deoxy-HbS, is reduced by greaterthan 80% in a binding assay.

In a further embodiment, the aptamers as described herein thatspecifically bind HbS are linked to conjugates to mediate intracellulardelivery into a cell of interest. Accordingly another embodiment of thepresently disclosed subject matter relates to compositions and methodsfor intracellular delivery of aptamers as described herein thatspecifically bind HbS into a cell of interest. “Cell of interest” asused herein refers to red blood cells (RBCs or erythrocytes) and includenucleated or non-nucleated adult and/or fetal red blood cells, but mayalso refer to erythroblasts, reticulocytes, and/or normoblasts. Suchconjugates that mediate intracellular delivery of the aptamers asdescribed herein that specifically bind HbS into a cell of interestinclude other aptamers that are known to specifically enter cells ofinterest (referred to herein as “delivery aptamers”) or other ligandsthat bind receptors on a cell of interest and are internalized by thecell (e.g., transferrin and transferrin receptors (CD71) on red bloodcells). Such conjugates may further include detectable labels such asfluorophores to facilitate methods of screening cells of interestcontaining the aptamers as described herein that specifically bind HbS.Where the conjugates are delivery aptamers, the delivery aptamers andthe aptamers as described herein that specifically bind HbS may belinked, for example, covalently or functionally through nucleic acidduplex formation. At least one of the linked aptamers may be partly orwholly comprised of 2′-modified RNA or DNA such as 2′F, 2′OH, 2′OMe,2′allyl, 2′MOE (methoxy-O-methyl) substituted nucleotides, and maycontain polyethylene glycol (PEG)-spacers and abasic residues. Covalentlinkages for delivery aptamers and other ligands may include, forexample, a linking moiety such as a nucleic acid moiety, a PNA moiety, apeptidic moiety, a disulfide bond or a polyethylene glycol (PEG) moiety.

III. Methods of Treatment

A. Methods for Treating Sickle Cell Disease

In one embodiment, the presently disclosed subject matter relates to amethod of treating or preventing sickle cell disease in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of a polynucleotide aptamer thatspecifically binds to sickle hemoglobin (HbS) and inhibitspolymerization of HbS. In a particular embodiment, the polynucleotideaptamer inhibits polymerization of HbS without a substantial orintolerable deleterious effect on hemoglobin's functional capabilities(e.g., a mild shift in Hb oxygen affinity might be associated with mildto moderate, but tolerable side effects).

As used herein, “sickle cell disease” means that the subject has atleast one sickle cell. As used herein, a “sickle cell” includes a cellwhich is an abnormal, crescent-shaped erythrocyte that contains sicklecell hemoglobin from a subject with sickle cell disease. “Sickling”includes the process whereby a normal-shaped cell becomescrescent-shaped. As described herein, sickle cell disease includes butis not limited to sickle cell anemia, sickle β-thalassemia, sicklecell-hemoglobin C disease and any other sickle hemoglobinopathy in whichHbS interacts with a hemoglobin other than HbS. “Sicklehemoglobinopathy” is an abnormality of hemoglobin which results insickle cell disease or sickle variants.

Any of the aptamers or modified aptamers described herein thatspecifically bind HbS and inhibit polymerization of HbS, includingoxy-HbS and/or deoxy-HbS, may be used within these methods of treatingsickle cell disease in a subject in need thereof. In one embodiment, thepolynucleotide aptamer that specifically binds to HbS and inhibitspolymerization of HbS for use within the methods for treating orpreventing sickle cell disease in a subject in need thereof is an RNAaptamer that comprises a nucleotide sequence that is at least 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to any one of SEQ ID NOS:2-60, or a fragment or analogthereof. In a particular embodiment, the polynucleotide aptamer thatspecifically binds to HbS and inhibits polymerization of HbS for usewithin the methods for treating or preventing sickle cell disease in asubject in need thereof is an RNA aptamer that comprises a nucleotidesequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS:2,4, 31, and 37, or, a fragment or analog thereof.

In some embodiments, the presently disclosed methods produce at leastabout a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or even 100% inhibition of polymerization ofHbS relative to polymerization of HbS measured in the absence ofaptamers or modified aptamers described herein that specifically bindHbS and inhibit polymerization of HbS, i.e., a control sample, in anassay.

In any of the above-described methods, the administering of any ofaptamers or modified aptamers described herein that specifically bindHbS and inhibit polymerization of HbS can result in at least about a10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or even 100% decrease in one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10) symptoms of sickle cell disease, compared to asubject that is not administered the aptamers or modified aptamersdescribed herein that specifically bind HbS and inhibit polymerizationof HbS.

In any of the above-described methods, the administering of the aptamersor modified aptamers described herein that specifically bind HbS andinhibit polymerization of HbS results in at least about a 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or even 100% decrease in the likelihood of developing sickle celldisease in a subject, compared to a control population of subjects thatare not administered the aptamers or modified aptamers described hereinthat specifically bind HbS and inhibit polymerization of HbS.

As used herein, the term “inhibit” or “inhibits” means to decrease,suppress, attenuate, diminish, arrest, or stabilize the development orprogression of a disease, disorder, or condition, the activity of abiological pathway, or a biological activity such as polymerization ofHbS, e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or even 100% compared to an untreated control subject, cell,biological pathway, or biological activity. By the term “decrease” ismeant to inhibit, suppress, attenuate, diminish, arrest, or stabilize asymptom of a sickle cell disease, disorder, or condition. Sickle celldisease associated symptoms include, but are not limited to, erythrocyte(RBC) sickling, oxygen release, increased HbS polymerization, hemolysis,tissue congestion and organ damage or dysfunction. It will beappreciated that, although not precluded, treating a disease, disorderor condition does not require that the disease, disorder, condition orsymptoms associated therewith be completely eliminated.

The method described above for treating or preventing sickle celldisease in a subject in need thereof may be carried out using a singleaptamer targeted to HbS, or may be carried out using two or moredifferent aptamers targeted to HbS, e.g., three, four, five, or sixdifferent aptamers.

For use within the methods for treating or preventing sickle celldisease in a subject in need thereof, the aptamers described herein thatspecifically bind to HbS and inhibit polymerization of HbS mayoptionally be administered in conjunction with other compounds (e.g.,therapeutic agents) or treatments (e.g., hydroxyurea or bloodtransfusions) useful in treating sickle cell disease. The othercompounds or treatments may optionally be administered concurrently. Asused herein, the word “concurrently” means sufficiently close in time toproduce a combined effect (that is, concurrently may be simultaneously,or it may be two or more events occurring within a short time periodbefore or after each other). The other compounds may be administeredseparately from the aptamers as disclosed herein, or may be combinedtogether with the aptamers as disclosed herein in a single composition.

As used herein, the terms “treat,” treating,” “treatment,” and the like,are meant to decrease, suppress, attenuate, diminish, arrest, theunderlying cause of a disease, disorder, or condition, or to stabilizethe development or progression of a disease, disorder, condition, and/orsymptoms associated therewith. The terms “treat,” “treating,”“treatment,” and the like, as used herein can refer to curative therapy,prophylactic therapy, and preventative therapy. Accordingly, as usedherein, “treating” means either slowing, stopping or reversing theprogression of the sickling of a cell, including reversing theprogression to the point of eliminating the presence of sickled cellsand/or reducing or eliminating the amount of polymerization ofhemoglobin, or the amelioration of symptoms associated with sickle celldisease. Sickle cell disease associated symptoms include, but are notlimited to, erythrocyte (RBC) sickling, oxygen release, increased HbSpolymerization, hemolysis, tissue congestion and organ damage ordysfunction. The treatment, administration, or therapy can beconsecutive or intermittent. Consecutive treatment, administration, ortherapy refers to treatment on at least a daily basis withoutinterruption in treatment by one or more days. Intermittent treatment oradministration, or treatment or administration in an intermittentfashion, refers to treatment that is not consecutive, but rather cyclicin nature. Treatment according to the presently disclosed methods canresult in complete relief or cure from a disease, disorder, orcondition, or partial amelioration of one or more symptoms of thedisease, disease, or condition, and can be temporary or permanent. Theterm “treatment” also is intended to encompass prophylaxis, therapy andcure.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disease, disorder, or condition in a subject, who doesnot have, but is at risk of or susceptible to developing a disease,disorder, or condition. Thus, in some embodiments, an agent can beadministered prophylactically to prevent the onset of a disease,disorder, or condition, or to prevent the recurrence of a disease,disorder, or condition.

The subject treated by the presently disclosed methods in their manyembodiments is desirably a human subject, although it is to beunderstood that the methods described herein are effective with respectto all vertebrate species, which are intended to be included in the term“subject.” Accordingly, a “subject” can include a human subject formedical purposes, such as for the treatment of an existing disease,disorder, condition or the prophylactic treatment for preventing theonset of a disease, disorder, or condition or an animal subject formedical, veterinary purposes, or developmental purposes. Suitable animalsubjects include mammals including, but not limited to, primates, e.g.,humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques andthe like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheepand the like; caprines, e.g., goats and the like; porcines, e.g., pigs,hogs, and the like; equines, e.g., horses, donkeys, zebras, and thelike; felines, including wild and domestic cats; canines, includingdogs; lagomorphs, including rabbits, hares, and the like; and rodents,including mice, rats, guinea pigs, and the like. An animal may be atransgenic animal. In some embodiments, the subject is a humanincluding, but not limited to, fetal, neonatal, infant, juvenile, andadult subjects. Further, a “subject” can include a patient afflictedwith or suspected of being afflicted with a disease, disorder, orcondition. Thus, the terms “subject” and “patient” are usedinterchangeably herein. Subjects also include animal disease models(e.g., rats or mice used in experiments, and the like).

B. Pharmaceutical Compositions

The presently disclosed pharmaceutical compositions and formulationsinclude pharmaceutical compositions of aptamers that specifically bindto HbS and inhibit polymerization of HbS as disclosed herein, alone orin combination with one or more additional therapeutic agents, inadmixture with a physiologically compatible carrier, which can beadministered to a subject, for example, a human subject, for therapeuticor prophylactic treatment. As used herein, “physiologically compatiblecarrier” refers to a physiologically acceptable diluent including, butnot limited to water, phosphate buffered saline, or saline, and, in someembodiments, can include an adjuvant. Acceptable carriers, excipients,or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and can include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid,BHA, and BHT; low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counter-ions such assodium; and/or nonionic surfactants such as Tween, Pluronics, or PEG.Adjuvants suitable for use with the presently disclosed compositionsinclude adjuvants known in the art including, but not limited to,incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide,and alum.

Compositions to be used for in vivo administration must be sterile,which can be achieved by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.Therapeutic compositions may be placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

In certain embodiments, the presently disclosed subject matter alsoincludes combination therapies. Additional therapeutic agents, which arenormally administered to treat or prevent sickle cell disease, may beadministered in combination with aptamers that specifically bind to HbSand inhibit polymerization of HbS as disclosed herein. These additionalagents may be administered separately, as part of a multiple dosageregimen, from the composition comprising aptamers that specifically bindto HbS and inhibit polymerization of HbS as disclosed herein.Alternatively, these agents may be part of a single dosage form, mixedtogether with the aptamers that specifically bind to HbS and inhibitpolymerization of HbS as disclosed herein, in a single composition.

By “in combination with” is meant the administration of a aptamers thatspecifically bind to HbS and inhibit polymerization of HbS as disclosedherein, with one or more therapeutic agents either simultaneously,sequentially, or a combination thereof. Therefore, a subjectadministered a combination of aptamers that specifically bind to HbS andinhibit polymerization of HbS as disclosed herein, can receive anaptamer that specifically binds to HbS and inhibits polymerization ofHbS as disclosed herein, and one or more therapeutic agents at the sametime (i.e., simultaneously) or at different times (i.e., sequentially,in either order, on the same day or on different days), so long as theeffect of the combination of both agents is achieved in the subject.When administered sequentially, the agents can be administered within 1,5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In otherembodiments, agents administered sequentially, can be administeredwithin 1, 5, 10, 15, 20 or more days of one another. Where the aptamerthat specifically binds to HbS and inhibits polymerization of HbS asdisclosed herein, and one or more therapeutic agents are administeredsimultaneously, they can be administered to the subject as separatepharmaceutical compositions, each comprising either an aptamer thatspecifically binds to HbS and inhibits polymerization of HbS asdisclosed herein, or one or more therapeutic agents, or be administeredto a subject as a single pharmaceutical composition comprising bothagents.

When administered in combination, the effective concentration of each ofthe agents to elicit a particular biological response may be less thanthe effective concentration of each agent when administered alone,thereby allowing a reduction in the dose of one or more of the agentsrelative to the dose that would be needed if the agent was administeredas a single agent. The effects of multiple agents may, but need not be,additive or synergistic. The agents may be administered multiple times.In such combination therapies, the therapeutic effect of the firstadministered agent is not diminished by the sequential, simultaneous orseparate administration of the subsequent agent(s).

C. Dosage and Mode of Administration

The presently disclosed pharmaceutical compositions can be administeredusing a variety of methods known in the art depending on the subject andthe particular disease, disorder, or condition being treated. Theadministering can be carried out by, for example, intravenous infusion;injection by intravenous, intraperitoneal, intracerebral, intramuscular,intraocular, intraarterial or intralesional routes; or topical or ocularapplication.

More particularly, as described herein, the presently disclosed aptamersthat specifically bind to HbS and inhibit polymerization of HbS can beadministered to a subject for therapy by any suitable route ofadministration, including orally, nasally, transmucosally, ocularly,rectally, intravaginally, parenterally, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intra-articular, intra-sternal,intra-synovial, intra-hepatic, intralesional, intracranial,intraperitoneal, intranasal, or intraocular injections,intracisternally, topically, as by powders, ointments or drops(including eyedrops), including buccally and sublingually,transdermally, through an inhalation spray, or other modes of deliveryknown in the art.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of an aptamer that specifically binds toHbS and inhibits polymerization of HbS, a compound, drug or othermaterial other than directly into the central nervous system, such thatit enters the patient's system and, thus, is subject to metabolism andother like processes, for example, subcutaneous administration.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intarterial, intrathecal,intracapsular, intraorbital, intraocular, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The presently disclosed pharmaceutical compositions can be manufacturedin a manner known in the art, e.g. by means of conventional mixing,dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping or lyophilizing processes.

More particularly, pharmaceutical compositions for oral use can beobtained through combination of an aptamer that specifically binds toHbS and inhibits polymerization of HbS with a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired, to obtaintablets or dragee cores. Suitable excipients include, but are notlimited to, carbohydrate or protein fillers, such as sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose, such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethyl cellulose; andgums including arabic and tragacanth; and proteins, such as gelatin andcollagen; and polyvinylpyrrolidone (PVP:povidone). If desired,disintegrating or solubilizing agents, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate, also can be added to the compositions.

Dragee cores are provided with suitable coatings, such as concentratedsugar solutions, which also can contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments can be added to the tablets ordragee coatings for product identification or to characterize thequantity of an aptamer that specifically binds to HbS and inhibitspolymerization of HbS, e.g., dosage, or different combinations ofaptamer doses.

Pharmaceutical compositions suitable for oral administration includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, e.g., a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain active ingredients admixedwith a filler or binder, such as lactose or starches, lubricants, suchas talc or magnesium stearate, and, optionally, stabilizers. In softcapsules, the aptamer that specifically binds to HbS and inhibitspolymerization of HbS can be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols(PEGs), with or without stabilizers. Stabilizers can be added aswarranted.

In some embodiments, the presently disclosed pharmaceutical compositionscan be administered by rechargeable or biodegradable devices. Forexample, a variety of slow-release polymeric devices have been developedand tested in vivo for the controlled delivery of drugs, includingproteinacious biopharmaceuticals. Suitable examples of sustained releasepreparations include semipermeable polymer matrices in the form ofshaped articles, e.g., films or microcapsules. Sustained releasematrices include polyesters, hydrogels, polylactides (U.S. Pat. No.3,773,919; EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., Biopolymers 22:547, 1983), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.15:167, 1981; Langer, Chem. Tech. 12:98, 1982), ethylene vinyl acetate(Langer et al., Id), or poly-D-(−)-3-hydroxybutyric acid (EP 133,988A).Sustained release compositions also include liposomally entrappedaptamers, which can be prepared by methods known per se (Epstein et al.,Proc. Natl. Acad. Sci. U.S.A. 82:3688, 1985; Hwang et al., Proc. Natl.Acad. Sci. U.S.A. 77:4030, 1980; U.S. Pat. Nos. 4,485,045 and 4,544,545;and EP 102,324A). Ordinarily, the liposomes are of the small (about200-800 Angstroms) unilamelar type in which the lipid content is greaterthan about 30 mol % cholesterol, the selected proportion being adjustedfor the optimal therapy. Such materials can comprise an implant, forexample, for sustained release of the presently disclosed aptamers thatspecifically bind to HbS and inhibit polymerization of HbS, which, insome embodiments, can be implanted at a particular, pre-determinedtarget site.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of aptamers that specifically bind to HbS and inhibitpolymerization of HbS. For injection, the presently disclosedpharmaceutical compositions can be formulated in aqueous solutions, forexample, in some embodiments, in physiologically compatible buffers,such as Hank's solution, Ringer's solution, or physiologically bufferedsaline. Aqueous injection suspensions can contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, suspensions of theaptamers that specifically bind to HbS and inhibit polymerization of HbSor vehicles include fatty oils, such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension also can contain suitable stabilizers oragents that increase the solubility of the aptamers that specificallybind to HbS and inhibit polymerization of HbS to allow for thepreparation of highly concentrated solutions.

For nasal or transmucosal administration generally, penetrantsappropriate to the particular barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

For inhalation delivery, the agents of the disclosure also can beformulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances such as, saline, preservatives, suchas benzyl alcohol, absorption promoters, and fluorocarbons.

Additional ingredients can be added to compositions for topicaladministration, as long as such ingredients are pharmaceuticallyacceptable and not deleterious to the epithelial cells or theirfunction. Further, such additional ingredients should not adverselyaffect the epithelial penetration efficiency of the composition, andshould not cause deterioration in the stability of the composition. Forexample, fragrances, opacifiers, antioxidants, gelling agents,stabilizers, surfactants, emollients, coloring agents, preservatives,buffering agents, and the like can be present. The pH of the presentlydisclosed topical composition can be adjusted to a physiologicallyacceptable range of from about 6.0 to about 9.0 by adding bufferingagents thereto such that the composition is physiologically compatiblewith a subject's skin.

In other embodiments, the pharmaceutical composition can be alyophilized powder, optionally including additives, such as 1 mM-50 mMhistidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5that is combined with buffer prior to use.

The presently disclosed subject matter also includes the use of aptamersthat specifically bind to HbS and inhibit polymerization of HbSdisclosed herein, in the manufacture of a medicament for sickle celldisease.

Regardless of the route of administration selected, the presentlydisclosed aptamers that specifically bind to HbS and inhibitpolymerization of HbS, which may be used in a suitable hydrated form,and/or the pharmaceutical compositions are formulated intopharmaceutically acceptable dosage forms such as described below or byother conventional methods known to those of skill in the art.

The term “effective amount,” as in “a therapeutically effective amount,”of a therapeutic agent refers to the amount of the agent necessary toelicit the desired biological response. As will be appreciated by thoseof ordinary skill in this art, the effective amount of an agent may varydepending on such factors as the desired biological endpoint, the agentto be delivered, the composition of the pharmaceutical composition, thetarget tissue or cell, and the like. More particularly, the term“effective amount” refers to an amount sufficient to produce the desiredeffect, e.g., to reduce or ameliorate the severity, duration,progression, or onset of a disease, disorder, or condition (e.g., adisease, condition, or disorder related to polymerization of HbS such assickle cell disease), or one or more symptoms thereof; prevent theadvancement of a disease, disorder, or condition, cause the regressionof a disease, disorder, or condition; prevent the recurrence,development, onset or progression of a symptom associated with adisease, disorder, or condition, or enhance or improve the prophylacticor therapeutic effect(s) of another therapy.

Actual dosage levels of the active ingredients in the presentlydisclosed pharmaceutical compositions can be varied so as to obtain anamount of the active ingredient that is effective to achieve the desiredtherapeutic response for a particular subject, composition, route ofadministration, and disease, disorder, or condition without being toxicto the subject. The selected dosage level will depend on a variety offactors including the activity of the particular aptamer employed, theroute of administration, the time of administration, the rate ofexcretion of the particular aptamer being employed, the duration of thetreatment, other drugs, aptamers and/or materials used in combinationwith the particular aptamer employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the aptamers that specifically bind to HbS and inhibitpolymerization of HbS, employed in the pharmaceutical composition atlevels lower than that required to achieve the desired therapeuticeffect and gradually increase the dosage until the desired effect isachieved. Accordingly, the dosage range for administration will beadjusted by the physician as necessary. It will be appreciated that anamount of an aptamer required for achieving the desired biologicalresponse, e.g., inhibition of polymerization of HbS, may be differentfrom the amount of compound effective for another purpose.

In general, a suitable daily dose of aptamers that specifically bind toHbS and inhibit polymerization of HbS, will be that amount of theaptamer that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. Generally, doses of the aptamers that specifically bindto HbS and inhibit polymerization of HbS will range from about 0.0001 toabout 1000 mg per kilogram of body weight of the subject per day. Incertain embodiments, the dosage is between about 1 μg/kg and about 500mg/kg, more preferably between about 0.01 mg/kg and about 50 mg/kg. Forexample, in certain embodiments, a dose can be about 1, 5, 10, 15, 20,or 40 mg/kg/day.

If desired, the effective daily dose of the aptamers that specificallybind to HbS and inhibit polymerization of HbS can be administered astwo, three, four, five, six or more sub-doses administered separately atappropriate intervals throughout the day, optionally, in unit dosageforms.

D. Kits or Pharmaceutical Systems

The presently disclosed aptamers that specifically bind to HbS andinhibit polymerization of HbS disclosed herein and compositions can beassembled into kits or pharmaceutical systems for use in treating orpreventing neurodegenerative diseases, disorders, or conditions. In someembodiments, the presently disclosed kits or pharmaceutical systemsinclude aptamers that specifically bind to HbS and inhibitpolymerization of HbS as disclosed herein. In particular embodiments,the aptamers that specifically bind to HbS and inhibit polymerization ofHbS as disclosed herein, are in unit dosage form. In furtherembodiments, the aptamers that specifically bind to HbS and inhibitpolymerization of HbS as disclosed herein, can be present together witha pharmaceutically acceptable solvent, carrier, excipient, or the like,as described herein.

In some embodiments, the presently disclosed kits comprise one or morecontainers, including, but not limited to a vial, tube, ampule, bottleand the like, for containing the compound. The one or more containersalso can be carried within a suitable carrier, such as a box, carton,tube or the like. Such containers can be made of plastic, glass,laminated paper, metal foil, or other materials suitable for holdingmedicaments.

In some embodiments, the container can hold a composition that is byitself or when combined with another composition effective for treatingor preventing the condition and may have a sterile access port (forexample the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle).Alternatively, or additionally, the article of manufacture may furtherinclude a second (or third) container including apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

The presently disclosed kits or pharmaceutical systems also can includeassociated instructions for using the aptamers that specifically bind toHbS and inhibit polymerization of HbS as disclosed herein for treatingor sickle cell disease. In some embodiments, the instructions includeone or more of the following: a description of the aptamer thatspecifically binds to HbS and inhibits polymerization of HbS asdisclosed herein; a dosage schedule and administration for treating orpreventing sickle cell disease; precautions; warnings; indications;counter-indications; overdosage information; adverse reactions; animalpharmacology; clinical studies; and references. The instructions can beprinted directly on a container (when present), as a label applied tothe container, as a separate sheet, pamphlet, card, or folder suppliedin or with the container.

IV. Antidotes

The presently disclosed subject matter also relates to antidotes for theaptamers that specifically bind to HbS and inhibit polymerization of HbSas disclosed herein. Such antidotes can comprise oligonucleotides thatare reverse complements of segments of the aptamers that specificallybind to HbS and inhibit polymerization of HbS as disclosed herein. Inaccordance with the presently disclosed subject matter, the antidote iscontacted with a targeted aptamer under conditions such that it binds tothe aptamer and modifies the interaction between the aptamer and itstarget molecule (e.g., HbS). Modification of that interaction can resultfrom modification of the aptamer structure as a result of binding by theantidote. The antidote can bind free aptamer and/or aptamer bound to itstarget molecule. In certain embodiments, the aptamer that specificallybinds to HbS and inhibits polymerization of HbS as disclosed herein isprovided in alternation with an antidote.

Antidotes of the presently disclosed subject matter can be designed soas to bind any particular aptamer with a high degree of specificity anda desired degree of affinity. The antidote can be designed so that uponbinding to the targeted aptamer, the three-dimensional structure of thataptamer is altered such that the aptamer can no longer bind to itstarget molecule or binds to its target molecule with less affinity.

Antidotes of the presently disclosed subject matter include anypharmaceutically acceptable agent that can bind an aptamer and modifythe interaction between that aptamer and its target molecule (e.g., bymodifying the structure of the aptamer) in a desired manner. Examples ofsuch antidotes include oligonucleotides complementary to at least aportion of the aptamers that specifically bind to HbS and inhibitpolymerization of HbS as disclosed herein (including ribozymes orDNAzymes or peptide nucleic acids), nucleic acid binding peptides,polypeptides or proteins including nucleic acid binding tripeptides (seegenerally, Hwang et al. (1999) Proc. Natl. Acad. Sci. USA 96:12997), andoligosaccharides such as aminoglycosides (see, generally, Davies et al.(1993) Chapter 8, p. 185, RNA World, Cold Spring Harbor LaboratoryPress, eds Gestlaad and Atkins; Werstuck et al. (1998) Science 282:296;U.S. Pat. Nos. 5,935,776 and 5,534,408; Chase et al. (1986) Ann. Rev.Biochem. 56:103; Eichhorn et al. (1968) J. Am. Chem. Soc. 90:7323; Daleet al. (1975) Biochemistry 14:2447; and Lippard et al. (1978) Acc. Chem.Res. 11:211).

Standard binding assays can be used to screen for antidotes of thepresently disclosed subject matter (e.g., using BIACORE assays).Candidate antidotes can be contacted with the aptamer to be targetedunder conditions favoring binding and a determination made as to whetherthe candidate antidote in fact binds the aptamer. Candidate antidotesthat are found to bind the aptamer can then be analyzed in anappropriate bioassay (which will vary depending on the aptamer and itstarget molecule) to determine if the candidate antidote can affect thebinding of the aptamer to its target molecule.

Where the antidote is an oligonucleotide, the antidote oligonucleotidedoes not need to be completely complementary to the aptamer thatspecifically binds to HbS and inhibits polymerization of HbS asdisclosed herein as long as the antidote sufficiently binds to orhybridizes to the aptamer to neutralize its activity. In one embodiment,the antidote of the presently disclosed subject matter is anoligonucleotide that comprises a sequence complementary to at least aportion of the targeted aptamer sequence. In one embodiment, theantidote oligonucleotide comprises a sequence complementary to up to 80,75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 consecutivenucleotides of the targeted aptamer.

V. Diagnostic Methods

In one embodiment, the presently disclosed subject matter provides amethod for diagnosing or predicting a sickle cell disease in a subjecthaving or at risk of developing a sickle cell disease or at risk ofpassing it on to offspring. The method includes contacting a biologicalsample from the subject with an aptamer that specifically binds to HbSand inhibits polymerization of HbS as disclosed herein.

The aptamers that specifically bind to HbS and inhibit polymerization ofHbS as disclosed herein are particularly well suited for diagnosticapplications. Aptamers represent a class of molecules that may be usedin place of antibodies for in vitro or in vivo diagnostic purposes. Theaptamers of the presently disclosed subject matter are thereforeparticularly useful as diagnostic reagents to detect the presence orabsence of the target substances to which they specifically bind, i.e.,HbS. Such diagnostic tests are conducted by contacting a biologicalsample with the specifically binding oligonucleotide to obtain a complexwhich is then detected by conventional means. For example, the aptamersmay be labeled using radioactive, fluorescent, or chromogenic labels andthe presence of label bound to solid support to which the targetsubstance has been bound through a specific or nonspecific binding meansdetected. Alternatively, the specifically binding oligonucleotides maybe used to effect initial complexation to the support. Means forconducting assays using such oligomers as specific binding partners willtrack those for standard specific binding partner based assays.

Accordingly, in one embodiment, the presently disclosed subject matterprovides a method for diagnosing or predicting a sickle cell disease ina subject having or at risk of developing a sickle cell disease or atrisk of passing it on to offspring, the method comprising: (a) obtaininga biological sample from the subject; (b) contacting the biologicalsample with a polynucleotide aptamer that specifically binds to HbS asdisclosed herein; and (c) detecting binding of the polynucleotideaptamer with HbS in the biological sample, wherein detection of bindingof the polynucleotide aptamer with HbS in the biological sample isindicative of the subject having or at risk of developing a sickle celldisease or at risk of passing it on to offspring. The aptamers can belabeled for detection using methods and labels known in the artincluding, but not limited to, fluorescent, luminescent, phosphorescent,radioactive, and/or colorimetric compounds.

As used herein, the phrase “biological sample” encompasses a variety ofsample types obtained from a subject and useful in the procedure of thepresently disclosed subject matter. In one embodiment of the presentlydisclosed subject matter, the biological sample comprises whole blood,hemocytes, serum, or plasma. However, biological samples may include,but are not limited to, solid tissue samples, liquid tissue samples,biological fluids, aspirates, cells and cell fragments. Specificexamples of biological samples include, but are not limited to, solidtissue samples obtained by surgical removal, pathology specimens,archived samples, or biopsy specimens, tissue cultures or cells derivedtherefrom and the progeny thereof, and sections or smears prepared fromany of these sources. Non-limiting examples of biological samplesinclude samples obtained from breast tissue, lymph nodes, and breasttumors. Biological samples also include any material derived from thebody of a vertebrate animal, including, but not limited to, blood,cerebrospinal fluid, serum, plasma, urine, nipple aspirate, fine needleaspirate, tissue lavage such as ductal lavage, saliva, sputum, ascitesfluid, liver, kidney, breast, bone, bone marrow, testes, brain, ovary,skin, lung, prostate, thyroid, pancreas, cervix, stomach, intestine,colorectal, brain, bladder, colon, nares, uterine, semen, lymph, vaginalpool, synovial fluid, spinal fluid, head and neck, nasopharynx tumors,amniotic fluid, breast milk, pulmonary sputum or surfactant, urine,fecal matter and other liquid samples of biologic origin.

VI. Capture Reagents

In another embodiment, the presently disclosed subject matter relates tothe use of aptamers that specifically bind to HbS as capture reagentsfor clearing clear HbS or other hemoglobins (including normalhemoglobin) from a biological sample. Within these methods of theinvention, the aptamers that specifically bind to HbS do not necessarilyneed to also inhibit polymerization of HbS.

Accordingly, in one embodiment the presently disclosed subject matter isdirected to a method of purifying hemoglobin from a biological samplecomprising providing a biological sample containing hemoglobin,contacting the biological sample with an aptamer that specifically bindsto HbS as disclosed herein under conditions effective to bind hemoglobinto the aptamer, and recovering the hemoglobin bound to the aptamer. Incertain embodiments, the hemoglobin is HbS. In other embodiments, thebiological sample comprises whole blood, hemocytes, serum, or plasma.

The methods for purifying hemoglobin from a biological sample mayinclude the use of a solid support comprising an immobilized aptamer.Thus, in one embodiment of the method of purifying hemoglobin from abiological sample, the step of contacting the biological sample with anaptamer that specifically binds to HbS as disclosed herein underconditions effective to bind hemoglobin to the aptamer comprisesproviding a solid support comprising an aptamer that specifically bindsto HbS as disclosed herein immobilized onto the solid support through aspacer.

As used herein, a “spacer” is intended to mean a molecule which isinserted between the aptamer and the solid support. Advantageously, thespacer is bound both to one end of the aptamer and to the solid support.Advantageously, such structure comprising a spacer does not immobilizedirectly the aptamer onto the solid support. The nature of the spacermay be chosen according to the knowledge of one skilled in the art; forexample the spacer may be a non specific oligonucleotide sequence or maybe polyethylene glycol (PEG). When the spacer is a non specificoligonucleotide sequence, said sequence may contain at least 5nucleotides, particularly between 5 and 15 nucleotides.

For immobilizing the aptamer onto a spacer, the aptamer may bechemically modified with various chemical groups such as groups enablingto covalently immobilize the aptamer, such as thiols, amines or anyother group that could react with chemical groups present on the supportor groups enabling to non-covalently immobilize the aptamer, such as thebiotin-streptavidin system. These techniques may also be used forimmobilizing the spacer onto the solid support.

Once immobilized onto the solid support via the spacer, the aptamer maybe modified at the free end thereof (i.e. the end that is not bound tothe spacer) by, without limitation, a chemically modified nucleotide(such as 2′ omethyl or 2′ fluoropyrimidine, 2′ ribopurine,phosphoramidite), a reversed nucleotide or a chemical group (PEG,polycations, cholesterol). These and other modifications to thepresently disclosed aptamers as disclosed elsewhere herein may be usedto protect the aptamer against enzymatic degradation.

The solid support may be an affinity chromatography column containing agel derived from agarose or cellulose or a synthetic gel such as anacrylamide, a methacrylate or a polystyrene derivative; a chip such as achip adapted for surface plasmon resonance; a membrane such as apolyamide, a polyacrylonitrile or a polyester membrane; a magnetic orparamagnetic bead.

VII. Rational Drug Design

In another embodiment, the presently disclosed subject matter relates tothe use of an aptamer that specifically binds to HbS and inhibitspolymerization of HbS as disclosed herein as a template for rationaldrug design.

For example, structures of RNA aptamers that recognize the shape of HbScould be determined by spectroscopy or X-ray crystallography. Thesestructures could be used to guide the rational design of drugs(mimetics) that would recognize and bind to HbS and inhibitpolymerization of HbS.

Accordingly, in one embodiment the presently disclosed subject matter isdirected to a method of using a three-dimensional structure of anaptamer that specifically binds to HbS and inhibits polymerization ofHbS as disclosed herein in a drug screening assay comprising:

(a) selecting a potential drug by performing rational drug design withthe three-dimensional structure of the polynucleotide aptamer thatspecifically binds to HbS and inhibits polymerization of HbS determinedfrom one or more sets of atomic coordinates; wherein said selecting isperformed in conjunction with computer modeling;

(b) contacting the potential drug with HbS;

(c) detecting the binding of the potential drug with the HbS; and

(d) detecting the inhibition of polymerization of HbS by the potentialdrug; wherein a potential drug is selected as a drug if the potentialdrug binds to HbS and inhibits polymerization of HbS.

Alternatively, a refined aptamer sequence can be elucidated by modifyinga known aptamer structure using software comprising “builder” typealgorithms which utilizes a set of atomic coordinates defining athree-dimensional structure of the binding pocket and thethree-dimensional structures of the known aptamer to computationallyassemble a refined aptamer. Ample guidance for performing rational drugdesign via software employing such “scanner” and “builder” typealgorithms is available in the literature of the art (e.g., Halperin etal. (2002) Proteins 47:409-43; Gohlke & Klebe (2001) Curr Opin StructBiol. 11:231-5; Zeng (2000) Comb. Chem. High Throughput Screen.3:355-62).

Criteria that may be employed by software programs used in rational drugdesign to qualify the binding of screened aptamer structures withbinding pockets and/or binding sites of HbS include gap space, hydrogenbonding, electrostatic interactions, van der Waals forces,hydrophilicity/hydrophobicity, etc. Generally, the greater the contactarea between the screened aptamer and the HbS binding region, the lowerthe steric hindrance, the lower the “gap space”, the greater the numberof hydrogen bonds, and the greater the sum total of the van der Waalsforces between the screened aptamer and the HbS binding region, thegreater will be the capacity of the screened aptamer to bind with thetarget HbS. The “gap space” refers to unoccupied space between the vander Waals surface of a screened aptamer positioned within a bindingpocket or site and the surface of the binding pocket or site defined byamino acid residues in the binding pocket or site. Gap space may beidentified, for example, using an algorithm based on a series of cubicgrids surrounding the docked molecule, with a user-defined grid spacing,and represents volume that could advantageously be occupied by amodifying the docked aptamer positioned within the binding region of theHbS.

Contact area between compounds may be directly calculated from thecoordinates of the compounds in docked conformation using the MS program(Connolly (1983) Science 221:709-713).

Suitable software employing “scanner” type algorithms include, forexample, docking software such as GRAM, DOCK, or AUTODOCK (reviewed inDunbrack et al. (1997) Folding and Design 2:27), AFFINITY software ofthe INSIGHTII package (Molecular Simulations Inc., 1996, San Diego,Calif.), GRID (Goodford (1985) J. Med. Chem. 28:849-857; GRID isavailable from Oxford University, Oxford, UK), and MCSS (Miranker &Karplus (1991) Proteins: Structure Function and Genetics 11:29-34; MCSSis available from Molecular Simulations, Burlington, Mass.).

The AUTODOCK program (Goodsell & Olson (1990) Proteins: Struct FunctGenet. 8:195-202; available from Scripps Research Institute, La Jolla,Calif.) helps in docking screened molecules to binding pockets in aflexible manner using a Monte Carlo simulated annealing approach. Theprocedure enables a search without bias introduced by the researcher.This bias can influence orientation and conformation of a screenedmolecule in the targeted binding pocket

The DOCK program (Kuntz et al. (1982) J. Mol. Biol. 161:269-288;available from University of California, San Francisco), is based on adescription of the negative image of a space-filling representation ofthe binding pocket, and includes a force field for energy evaluation,limited conformational flexibility and consideration of hydrophobicityin the energy evaluation.

Modeling or docking may be followed by energy minimization with standardmolecular mechanics force fields or dynamics with programs such asCHARMM (Brooks et al. (1983) J. Comp. Chem. 4:187-217) or AMBER (Weineret al. (1984) J. Am. Chem. Soc. 106:765-784). As used herein,“minimization of energy” means achieving an atomic geometry of achemical structure via systematic alteration such that any further minorperturbation of the atomic geometry would cause the total energy of thesystem as measured by a molecular mechanics force-field to increase.Minimization and molecular mechanics force fields are well understood incomputational chemistry (e.g., Burkert & Allinger, “MolecularMechanics”, ACS Monograph 177, pp. 59-78, American Chemical Society,Washington, D.C. (1982)).

Programs employing “builder” type algorithms include LEGEND (Nishibata &Itai (1991) Tetrahedron 47:8985; available from Molecular Simulations,Burlington, Mass.), LEAPFROG (Tripos Associates, St. Louis, Mo.), CAVEAT(Bartlett et al. (1989) Special Pub Royal Chem Soc. 78:182-196;available from University of California, Berkeley), HOOK (MolecularSimulations, Burlington, Mass.), and LUDI (Bohm (1992) J. Comp. AidMolec. Design 6:61-78; available from Biosym Technologies, San Diego,Calif.).

The CAVEAT program suggests binding molecules based on desired bondvectors. The HOOK program proposes docking sites by using multiplecopies of functional groups in simultaneous searches. LUDI is a programbased on fragments rather than on descriptors which proposes somewhatlarger fragments to match with a binding pocket and scores its hitsbased on geometric criteria taken from the Cambridge Structural Database(CSD), the Protein Data Bank (PDB) and on criteria based on bindingdata. LUDI may be advantageously employed to calculate the inhibitionconstant of a docked chemical structure Inhibition constants (Ki values)of compounds in the final docking positions can be evaluated using LUDIsoftware.

During or following rational drug design, docking of an intermediatechemical structure or of an aptamer with the HbS binding pocket or sitemay be visualized via structural models, such as three-dimensionalmodels, thereof displayed on a computer screen, so as to advantageouslyallow user intervention during the rational drug design to optimize achemical structure.

Software programs useful for displaying such three-dimensionalstructural models, include RIBBONS (Carson (1997) Methods in Enzymology277:25), O (Jones et al. (1991) Acta Crystallogr. A47:110), DINO; andQUANTA, INSIGHT, SYBYL, MACROMODE, ICM, MOLMOL, RASMOL and GRASP(reviewed in Kraulis (1991) Appl Crystallogr. 24:946).

Other molecular modeling techniques may also be employed in accordancewith the presently disclosed subject matter (e.g., Cohen et al. (1990)J. Med. Chem. 33:883-894; Navia & Murcko (1992) Current Opinions inStructural Biology 2:202-210). For example, where the structures of testcompounds are known, a model of the test compound may be superimposedover the model of the structure of the aptamers as disclosed herein.Numerous methods and techniques are known in the art for performing thisstep, any of which may be used (e.g., Farmer “Drug Design”, Ariens(ed.), Vol. 10, pp 119-143 (Academic Press, New York, 1980); U.S. Pat.No. 5,331,573; U.S. Pat. No. 5,500,807; Verlinde (1994) Structure2:577-587; and Kuntz (1992) Science 257:1078-108).

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, parameters,quantities, characteristics, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about” even though the term “about” may notexpressly appear with the value, amount or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are not and need not beexact, but may be approximate and/or larger or smaller as desired,reflecting tolerances, conversion factors, rounding off, measurementerror and the like, and other factors known to those of skill in the artdepending on the desired properties sought to be obtained by thepresently disclosed subject matter. For example, the term “about,” whenreferring to a value can be meant to encompass variations of, in someembodiments, ±100% in some embodiments ±50%, in some embodiments ±20%,in some embodiments ±10%, in some embodiments ±5%, in some embodiments±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed methods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Example 1

The primary aim of the present study was to generate one or more RNAaptamers that would bind sickle hemoglobin (HbS) in such a way thatpolymerization of HbS would be inhibited, without a deleterious effecton hemoglobin's functional capabilities. Preventing the polymerizationof HbS would essentially cure sickle cell anemia, since thecomplications of the disease arise directly as a result of red bloodcell changes brought about by HbS polymerization. Because it is thedeoxygenated form of HbS that polymerizes, the creation andcharacterization of aptamers to deoxyHbS were conducted first. However,aptamers to oxyHbS were also created and characterized in an effort tocast a large net and limit assumptions about which type of aptamers maybe more effective ultimately in preventing polymerization. Additionally,aptamers that specifically bind HbS but do not have any effect onpolymerization might be useful as reagents for other scientific studies.For example, these could be used to clear HbS or other hemoglobins(including normal hemoglobin) from plasma for proteomic studies or beused to identify the presence of HbS in a patient sample, or fortherapeutics.

To accomplish these goals, the “systematic evolution of ligands byexponential enrichment” (SELEX) process was used to select for aptamersthat bind specifically to HbS in either its oxygenated or deoxygenatedstates. Each aptamer pool was analyzed for its ability to bind bothoxy-HbS and deoxy-HbS at different rounds in the selection process.However, both positive and negative selection were used, includingexperiments where the bound aptamers was recovered from the target, andexperiments where the aptamers that recognized more than one target wereremoved from the pool (for example, by absorbing pools of molecules thatbind both HbA and HbS to HbA, allowing the molecules that bind only to Sto “flow through.”) Once a relatively small pool of high affinityaptamers was obtained, individual aptamers were then amplified andtested for their ability to inhibit polymerization in a closed anaerobicsystem, in which sodium dithionite was used to deoxygenate hemoglobin.

Materials and Methods

Preparation of Hemoglobin

After obtaining informed consent, venous blood was drawn from anuntransfused patient with homozygous SS disease. Red cells were washed 5times with PBS, hemolyzed in 3.5 volumes of distilled water, andstromata were removed by centrifugation at 20,000 g for 25 minutes.Hemoglobin-rich extract was dialyzed into 0.05M tris-Cl, pH 8.3, andpurified HbS was obtained by separation on a DEAE Sephadex A-50 anionexchange column, developing with a gradient of 0.05M Tris-HCl, pH 8.3 to0.05M Tris-HCl pH 7.3. The HbS was dialyzed against 2 mM HEPES, pH 7.4for the SELEX process and against 1M potassium phosphate buffer, pH 7.1for use in the polymerization assays, and stored in small aliquots at−80° C. Hemoglobin concentrations were determined by standard methods.When necessary, hemoglobin was concentrated with Amicon Ultra 30Kcentrifugal filters (Millipore, Billerica, Mass.). Proportions ofhemoglobins at different oxidation states within a solution weredetermined by the method of Benesch, Benesch and Yung (Benesch, Benesch,& Yung (1973) Anal Biochem. 55:245-248).

Selection of Aptamers Through Systematic Evolution of Ligands byExponential Enrichment (SELEX)

The initial oligonucleotide library was the Sel2 library (TrilinkBiotechnologies, San Diego, Calif.) comprising the sequence5′-GGGAGGACGAUGCGG(N₄₀)—CAGACGACUCGCUGAGGAUCCGAGA-3′ (SEQ ID NO:1),where N₄₀ represents a random sequence of 40 nucleotides. Template DNAwas synthesized with the Klenow fragment of DNA polymerase I (NewEngland Biolabs, Ipswich, Mass.). Two separate selections wereperformed. Both selections shared the first four rounds, which werecarried out as described below, using deoxygenated HbS as the targetprotein. For these rounds, binding was carried out at 37° for 5 minutes.After round 4, the resulting RNA pool was utilized to continue with twoseparate selections. The first targeted HbS in its oxygenated state.Measurements of the oxidation states of HbS in a freshly-thawed aliquotshowed approximately 83-95% to be oxygenated hemoglobin, so freshlythawed HbS was used in the binding steps. Incubation was carried out atroom temperature for 10 minutes at a ratio of 5 moles RNA per mole ofprotein in round 5, increasing to 9 moles RNA per mole of protein byround 14. Bound RNA was collected by capturing the protein on anitrocellulose membrane, eluting and extracting the RNA. RT-PCRreactions were performed on the eluted RNA with AMV ReverseTranscriptase (Roche, Indianapolis, Ind.) and Platinum Taq Polymerase(Invitrogen, Grand Island, N.Y.). Transcription was performed with theDurascribe T7 kit (Epicentre, Madison, Wis.), which incorporates themodified nucleotides 2′-fluorine-dCTP and 2′-fluorine-dUTP into thesequences to generate nuclease-resistant RNA.

The second selection targeted deoxygenated HbS. Paired rounds wereperformed in which cycles of selection against deoxy-HbS were alternatedwith cycles of subtractive binding with oxy-HbS, described below. Inorder to deoxygenate the HbS prior to incubation, it was thawed, exposedto a vacuum by injection into a vacuum tube with a septum cap, androcked at room temperature for 1 hour. The hemoglobin was then removedfrom the tube, and incubation with RNA aptamer library was immediatelycarried out at room temperature for 10 minutes at a ratio of 3 moles RNAper mole of protein in round 1 then stepwise up to 7 moles RNA per moleprotein by round 9. For subtractive binding, the oligo pool wasincubated with oxy-HbS at room temperature for 10 minutes and theunbound flow-through material was collected and saved, following proteincapture on a nitrocellulose membrane. Butanol extraction was employed toconcentrate the unbound oligos. Subtractive binding followed by standardbinding was done for multiple “paired” rounds, with RNA ratios for theoxy-HbS rounds similar to those for the deoxy-HbS rounds, except for thefinal cycle of subtractive binding, in which the ratio was 5 moles RNAper mole protein. This lower ratio allowed the oxygenated protein tobind and retain a higher fraction of oxy-HbS-targeted aptamers in thepool. Oxy-HbS oligo selection rounds 1-7 and deoxy-HbS oligo selectionrounds 1-5 were performed in low salt binding buffer (20 mM HEPES pH7.4, 50 mM NaCl, 2 mM CaCl₂, 0.01% BSA) and low salt wash buffer (20 mMHEPES pH 7.4, 50 mM NaCl, 2 mM CaCl₂). Oxy-HbS selection rounds 8-14 anddeoxy-HbS selection rounds 6-9 were performed in high salt bindingbuffer (20 mM HEPES pH 7.4, 150 mM NaCl, 2 mM CaCl₂, 0.01% BSA) and highsalt wash buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 2 mM CaCl₂). The lastcycle of subtractive binding for deoxy-HbS aptamers, using oxy-HbS asthe target, was done in low salt buffers, again to decrease thestringency of aptamer-protein binding.

Binding Assays

Aptamers were dephosphorylated with bacterial alkaline phosphatase(Invitrogen, Grand Island, N.Y.) and 5′ end-labelled with α-³²P-ATP(Perkin Elmer, Waltham, Mass.) using T₄ polynucleotide kinase (NewEngland Biolabs, Ipswich, Mass.). RNA was diluted to 2,000 cpm/ul in theappropriate binding buffer and heated at 65° C. for 5 minutes. For theoligomer pool targeting oxyHbS, the hemoglobin was thawed and used tomake a dilution series in binding buffer. For the oligo pool targetingdeoxyHbS the hemoglobin was thawed, exposed to a vacuum and rocked atroom temperature for 1 hour, and used immediately to make a dilutionseries in binding buffer. For both assays, 5 ul of labeled RNA (10,000cpm) was added to each tube of the dilution series, such that the finalprotein concentrations ranged from 0.078 uM to 10 uM, in 2-foldincrements. Each mixture was incubated (at 37° C. for 5 minutes forrounds 0 and 4, and at room temperature for 10 minutes for subsequentrounds) and passed over a nitrocellulose membrane to capture the HbS andbound RNA, with the non-bound RNA captured on a nylon membrane. Thefractions of bound and unbound RNA were quantified with a BeckmanLS-3801 scintillation counter, and the nonspecific binding subtracted.

Cloning, Sequencing and RNA Preparation

At rounds 7 and 9 of selection targeting deoxy-HbS and rounds 11 and 14of selection targeting oxy-HbS, cDNA was synthesized from RNA and usedfor cloning and sequencing of individual aptamers. DNA oligos wereligated into the pCR2.1-TOPO vector using the TOPO cloning kit(Invitrogen, Grand Island, N.Y.) and T₄ DNA ligase (New England Biolabs,Ipswich, Mass.). Transformations were performed using One Shot TOP 10cells (Invitrogen, Grand island, NY), and following overnight growth,mini-preps were performed with the Qiaprep spin miniprep kit (Qiagen,Valencia, Calif.) to produce DNA for sequencing. Sequencing was carriedout at the Johns Hopkins Genetic Resources Core Facility. Largequantities of aptamer for analysis were generated from clone DNA bytranscription using the Durascribe T7 kit (Epicentre, Madison, Wis.).RNA was purified by running on a 12% denaturing gel and eluted in dH₂O.

Polymerization Assays

Sickle hemoglobin in 1 M potassium phosphate buffer, pH 7.1 was thawedon ice, concentrated in an Amicon Ultra 30K centrifugal filter tube(Millipore, Billerica, Mass.) at 4° C. and kept on ice. Aptamers indistilled H₂O or H₂O alone as a control were thawed on ice, aliquotedinto tubes for each individual replicate, heated at 65° C. for 5minutes, and placed on ice. SM20, a non-related aptamer targeting theplasminogen activator inhibitor-1 (PAI-1) and possessing flankingsequences identical to those generated here, was used as a negativeaptamer control. To deoxygenate sodium dithionite powder (Sigma, St.Louis, Mo.), it was placed in a tube with a rubber septum cap andflushed with nitrogen gas by inserting one needle for gas inlet and oneneedle for gas outlet into the cap (Adachi & Asakura (1979) J. Biol.Chem. 254:7765-7771). A buffer solution of 1.6 M potassium phosphatebuffer pH 7.8 was deoxygenated similarly in separate tubes flushed withnitrogen gas. The dithionite powder and buffer were exposed to nitrogenfor 1½ hours, flushing with new nitrogen gas every ½ hour, then placedon ice. Sodium dithionite stock solution and subsequent dilutions werethen made using a Hamilton gas-tight syringe to transfer buffer from onetube to another (Adachi & Asakura (1979) J. Biol. Chem. 254:7765-7771).Each replicate was run in a closed 1 cm quartz cuvette (Starna Cells,Atascadero, Calif.) that had been flushed with nitrogen gas through aseptum cap as described above, and placed on ice. Cold 1.6 M potassiumphosphate buffer pH, 7.8 and HbS were added to the tube containingaptamer, mixed well, and added to the nitrogen-filled cuvette using aHamilton gas-tight syringe. The syringe was immediately washed and usedto transfer sodium dithionite solution from its dilution tube into thecuvette. The final concentrations of all components in the cuvette were0.12 mM HbS, 0.48 mM sodium dithionite, and 0.01 mM aptamer in 1.49 Mpotassium phosphate, pH 7.9. After addition of dithionite, the contentswere briefly mixed and put in a 37° C. water bath. The cuvette wasremoved one minute after incubation began and turbidity measured with aBeckman DU-640B spectrophotometer (Beckman Coulter, Inc., Brea, Calif.)at a wavelength of 700 nm (Adachi & Asakura (1979) J. Biol. Chem.254:7765-7771; Harrington (1998) Comp. Biochem. Physiol.119B(2):305-309; Eaton & Hofrichter (1987) Blood 70:1245-1266; Knee &Mukerji (2009) Biochemistry 48:9903-9911; Magdoff-Fairchild et. al.(1976) Proc. Nat. Acad. Sci. 73(4):990-994). Measurements were alsotaken at 540, 560 and 576 nm in order to calculate the fractions of HbSin various oxygenation states. After the spectrophotometric measurement,the cuvette was immediately returned to 37° C. Subsequent readings weredone in a similar manner every 3 minutes through the 34 minute timepoint, then every 5 minutes from the 40 minute to the 70 minute timepoints.

Results

Different Sets of Aptamers Generated Targeting Oxy-HbS Versus Deoxy-HbS

One of the goals of the present study was to generate aptamers that bindto HbS in either its oxygenated or deoxygenated states specifically andfurther to identify aptamers that when bound result in inhibition ofpolymerization of deoxy-HbS. While aptamers binding selectively to oxy-or deoxyhemoglobin were targeted, it was also recognized that aptamersbinding to both oxygenated and deoxygenated Hb would likely beidentified and would be of value, potentially inhibiting HbSpolymerization as well. Clones were obtained and sequenced at rounds 11and 14 of the oxy-HbS aptamer selection and rounds 7 and 9 of thedeoxy-HbS aptamer selection. 57 total aptamers were identified thatbound to deoxy-HbS. Of these, several aptamers were represented multipletimes; many sequences were represented only once. 15 total aptamers wereidentified that bound oxy-HbS.

The unique sequences for the identified clones are shown in Table 1.Only one of the clones is represented in both the oxy and deoxy aptamerpool. Several aptamers identified in the group targeting deoxy-HbS werefurther amplified and analyzed for their ability to inhibitpolymerization. Although the remainder of the discussion below appliesto these deoxy-HbS aptamers that have been partially characterized fortheir anti-polymerization activity, all clones selected against bothdeoxy and oxy hemoglobin may potentially inhibit HbS polymerization aswell.

TABLE 1 Complete Aptamer Sequences(unique or representative of an identical familyDeoxyHbS Aptamer Sequences Clone deoxy 1 (SEQ ID NO: 2) 5′GGGAGGACGAUGCGGccgauuagaacugggcugcgaucggagauccucuagguuuCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 2 (SEQ ID NO: 3) 5′GGGAGGACGAUGCGGgccgagggauucgguguagacucugcacaguccugaaaagCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 3-A (SEQ ID NO: 4) 5′GGGAGGACGAUGCGGccgauuagaacugggcugaggcguucugcauuucggugauCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 3-B (SEQ ID NO: 5) 5′GGGAGGACGAUGCGGccgauuagaacugggcuguuccgacucugcauccggugauCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 5 (SEQ ID NO: 6) 5′GGGAGGACGAUGCGGuuggugaagggaggucagcauaucuucccgcgggaagcgaCGGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 7-A (SEQ ID NO: 7) 5′GGGAGGACGAUGCGGauccacggguaagggugagggacgacaucaaggcgagauuCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 8 (SEQ ID NO: 8) 5′GGGAGGACGAUGCGGuacgauuagaacuggugccgaacagcgcucguugaagacaCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 9 (SEQ ID NO: 9) 5′GGGAGGACGAUGCGGaggaaguaggguucguccauugggcgaguggccuguguuaCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 10 (SEQ ID NO: 10) 5′GGGAGGACGAUGCGGcacgguauaguggaguggguaggcaucgcucgacgagugaCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 15 (SEQ ID NO: 11) 5′GGGAGGACGAUGCGGgaguagggagguaaucgccaccccaacgcggagacagcgaCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 19-C-1 (SEQ ID NO: 12)5′ GGGAGGACGAUGCGGucgauagggggacggaccgcgcuggaaacucaacguagcaCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 20 (SEQ ID NO: 13) 5′GGGAGGACGAUGCGGcacugaugggaguuggaucagugucgagcgguaucugcagCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 22 (SEQ ID NO: 14) 5′GGGAGGACGAUGCGGgaguagggagguaaucgucaccccaacgcggagacagcgaCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 24 (SEQ ID NO: 15) 5′GGGAGGACGAUGCGGaagcauacaguuuagugugcuagggugggacucagugauCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 28-A (SEQ ID NO: 16) 5′GGGAGGACGAUGCGGuccuacuuuccccaauuuguaacagcucuccgcacagcagCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 30 (SEQ ID NO: 17) 5′GGGAGGACGAUGCGGcgguguagggaucgucagucucggaaugaccucacagaagCAGACGACUCGGUGAGGAUCCGAGA 3′ Clone deoxy 31 (SEQ ID NO: 18) 5′GGGAGGACGAUGCGGccagcaggaggaugggugccgcacucggauauucacguguCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 33-A (SEQ ID NO: 19) 5′GGGAGGACGAUGCGGgacuaagcacaacucaacuagaacgaaccuauuccaucauCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 34-D (SEQ ID NO: 20) 5′GGGAGGACGAUGCGGaacggaggaguguccucucagcugacagucgugcauacuaCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 37-A (SEQ ID NO: 21) 5′GGGAGGACGAUGCGGaacucgauccaucaucgugacugcguacgugucaacuaagCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 40 (SEQ ID NO: 22) 5′GGGAGGACGAUGCGGgacggucauagagccggccgacauuagagccgggaauccaCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 44-A (SEQ ID NO: 23) 5′GGGAGGACGAUGCGGuggagaggggaaucguccugcgcacucugucuccugagagCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 45 (SEQ ID NO: 24) 5′GGGAGGACGAUGCGGuguauccgccaguaugauuaacaucuauaagucccuauguCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 46 (SEQ ID NO: 25) 5′GGGAGGACGAUGCGGcuaaccuuguuagggccccauacagcaucgagugacggauCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 47 (SEQ ID NO: 26) 5′GGGAGGACGAUGCGGugcacaggaggugguacacugcgcucgauucaucagcgcaCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 48 (SEQ ID NO: 27) 5′GGGAGGACGAUGCGGcaugugagggaggagguccgcgucauaaacuccaggaccaCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 50 (SEQ ID NO: 28) 5′GGGAGGACGAUGCGGaagcaauagcucgccguacaguuguccugccgcucguguuCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone deoxy 52 (SEQ ID NO: 29) 5′GGGAGGACGAUGCGGgaguagggagguaagcaguggacuaacgagauucggugagCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone EMdeoxy 8 (SEQ ID NO: 30) 5′GGGAGGACGAUGCGGcgagcaaccggaacucggcucuuaugaccagccaacuuaaCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone EMdeoxy 8-A (SEQ ID NO: 31)5′ GGGAGGACGAUGCGGcgagcaaccugaacucggcuauuaggaccagccaacuuaaCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone EMdeoxy 11 (SEQ ID NO: 32) 5′GGGAGGACGAUGCGSgaucggaaccagcgugacgacgcgcggaucaacuccggugCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone EMdeoxy 11-A (SEQ ID NO: 33)5′ GGGAGGACGAUGCGGgaucggaaccagcgugacgaagcgcggaucaacuccggugCUGACGACUCGCUGAGGAUCCGAGA 3′ Clone EMdeoxy 12 (SEQ ID NO: 34) 5′GGGAGGACGAUGCGGccgauuagaacugggucccgcuguacccuagggaucgaCAGACGACUCGCUGAGGAUCCGAGA 3′ OXyHbS Apt-Inner SequencesClone oxy 1 (SEQ ID NO: 35) 5′GGGAGGACGAUGCGGagacccaagcgccacgucuggcaugugagggaggagguacCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone oxy 2 (SEQ ID NO: 36) 5′GGGAGGACGAUGCGGagagccaagcgccacgucuggcaugugaggggggagguacCAGACGACUCGGUGAGGAUCCGAGA 3′ Clone oxy 3-8 (SEQ ID NO: 37) 5′GGGAGGACGAUGCGGaaacucaucgguagccuuccugcggucagucuauuaggacCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone oxy 4-8 (SEQ ID NO: 38) 5′GGGAGGACGAUGCGGcaauuaccucagccucccuagacacgucgucuauuaggacCAGACGACUCGGUGAGGAUCCGAGA 3′ Clone oxy 5-A (SEQ ID NO: 39) 5′GGGAGGACGAUGCGGcagucuuccgguaagcacggaggugaggggagcuuagcguCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone oxy 6 (SEQ ID NO: 40) 5′GGGAGGACGAUGCGGauaugccaugggucgcucgagugaggucgucuauuaggacCAGACGACUCGGUGAGGAUCCGAGA 3′ Clone oxy 7 (SEQ ID NO: 41) 5′GGGAGGACGAUGCGGagagccaagcgccacgucuggcaugugagggaggagguacCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone oxy 8 (SEQ ID NO: 42) 5′GGGAGGACGAUGCGGauuggcgcuauuaggaccagcuccguccgcaacuggucccGAGACGACUCGCUGAGGAUCCGAGA 3′ Clone ocy 9 (SEQ ID NO: 43) 5′GGGAGGACGAUGCGGgaacagacccauggcaaucucgcgacgucuucggccgcugCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone oxy 10 (SEQ ID NO: 44) 5′GGGAGGACGAUGCGGuacaacagguucauacggcgcguuguuccuuggcugacgCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone oxy 11 (SEQ ID NO: 45) 5′GGGAGGACGAUGCGGcacuauuaggaccagcgccuguugucucgauaagcuccgcCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone oxy 12 (SEQ ID NO: 46) 5′GGGAGGACGAUGCGGauuggcgcuauuaggaccagcuccguccgcaacugaucccGAGACGACUCGCUGAGGAUCCGAGA 3′ Clone oxy 13-A (SEQ ID NO: 47) 5′GGGAGGACGAUGCGGcuauuaggaccagccguguagaauucguagcgaugugacgCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone oxy 13-B (SEQ ID NO: 48) 5′GGGAGGACGAUGCGGuucgcgcuauuaggaccagugcgaacguggguauacauguCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone EMoxy 2-B (SEQ ID NO: 49) 5′GGGAGGACGAUGGGGaacacacgggacgagccuggcgguugucgccuauuaggacCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone EMoxy 3 (SEQ ID NO: 50) 5′GGGAGGACGAUGCGGguccaugcuuuaaacugcaauuucccguuuacacgggcuguCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone EMoxy 3-M (SEQ ID NO: 51) 5′GGGAGGACGAUGCGGaccaccgaaucacgaggugcgagacauugguuccccgccgCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone EMoxy 4 (SEQ ID NO: 52) 5′GGGAGGACGAUGCGGgggacaauaguccacgacuacaugucggugcgucggagguCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone EMoxy 6-A (SEQ ID NO: 53) 5′GGGAGGACGAUGCGGcuauuaggaccagcugccaauguuaagucuaccccagcagCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone EMoxy 6-C (SEQ ID NO: 54) 5′GGGAGGACGAUGCGGcuuacguauggucacggaggugugggggaacauacagcagCAGACGACUCGGUGAGGAUCCGAGA 3′ Clone EMoxy 8 (SEQ ID NO: 55) 5′GGGAGGACGAUGCGGuuggugaccuauucaggcguaggcauauaaacuacgaggcCAGACGACUCGGUGAGGAUCCGAGA 3′ Clone EMoxy 8 (SEQ ID NO: 56) 5′GGGAGGACGAUGCGGcuauuaggaccagcugccaauguuaagucuaccccagcggCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone EMoxy 11 (SEQ ID NO: 57) 5′GGGAGGACGAUGCGGgcacgacacgccgauuagaacugggcgaucuuggucgagcCAGACGACUCGGUGAGGAUCCGAGA 3′ Clone EMoxy 12 (SEQ ID NO: 58) 5′GGGAGGACGAUGCGGcgauacgaccgcaugaguauaccgucgugcuucccggcugCAGACGACUCGCUGAGGAUCCGAGA 3′ Clone EMoxy 13 (SEQ ID NO: 59) 5′GGGAGGACGAUGCGGauuggcgcuauuaggaccagcuccguccgcaaccggucccCAGACGACUCGGUGAGGAUCCGAGA 3′ Clone EMoxy 14 (SEQ ID NO: 60) 5′GGGAGGACGAUGCGGauuggcgcuauuaggaccagcuccguccgcaacuggucccCAGACGACUCGCUGAGGAUCCGAGA 3′

Aptamer Pools Generated that Bind Specifically to Deoxy-HbS

Binding of the deoxy HbS-selected aptamers to oxy- and deoxy-hemoglobinwas assessed at various rounds of selection. FIG. 1 shows that by theend of the selection process, the aptamers selected against deoxyhemoglobin bind to deoxy hemoglobin in a dose dependent saturablemanner.

Aptamer Deoxy-3-A Inhibits Polymerization of Deoxygenated HbS

In order to evaluate individual aptamers' ability to inhibitpolymerization, aptamers were amplified and added to an anaerobicsolution of deoxygenated HbS, in which spectrophotometric measurementsof turbidity at a wavelength of 700 nm reflected the extent ofpolymerization. It was necessary to develop a system in which HbS wouldreliably and reproducibly polymerize using the smallest possibleconcentration in order to minimize the quantities of aptamer necessaryfor testing. In HbS polymerization studies, shorter lag times and higherpolymerization rates are associated with higher temperature, higherhemoglobin concentration, the use of potassium phosphate buffer, andhigher buffer concentrations; therefore, in order to enhancepolymerization in a low concentration HbS system, we utilized a buffersolution of 1.49 M potassium phosphate buffer and conducted theincubations at 37° C. Sodium dithionite at a concentration of 0.48 mMwas used to deoxygenate HbS. Test assays (data not shown) showed thatthis concentration of sodium dithionite in our system consistentlyresulted in solutions of 93-96% deoxygenated HbS.

Each aptamer was tested at a concentration of 0.01 mM (an aptamer toheme molar ratio of 1:12). From the initial set of activity assays, anaptamer denoted deoxy-3-A (SEQ ID NO:4) was identified as causingreduced polymerization, with increased lag times and decreased maximalpolymerization over the time frame of the experiment (FIG. 2). This isproof of principle that an RNA aptamer can alter the polymerization ofHbS, and shows that this agent could be useful as a therapy for sicklecell anemia. It also describes a specific, unique, but modifiableaptamer reagent that has this property. FIG. 2 summarizes data for 4separate experiments with the deoxy 3-A aptamer (SEQ ID NO:4),demonstrating consistent inhibition of HbS polymerization in multipleruns as compared to a control aptamer (SM20) or water control.Preliminary results from two additional aptamers, Deoxy-1 (SEQ ID NO:2)and Deoxy EM8-A (SEQ ID NO:31), have shown some inhibition ofpolymerization as well (FIGS. 3 and 4). FIG. 5 summarizes data for theOxy 3-B aptamer (SEQ ID NO:37), demonstrating consistent inhibition ofHbS polymerization in multiple runs as compared to water control.

To determine if deoxy 3-A inhibited polymerization of HbS in adose-dependent manner, HbS polymerization studies were performed aspreviously described in the presence of increasing concentrations of thedeoxy-3A aptamer from 0.3125 micromolar to 10 micromolar finalconcentration. Water only controls as well as non-specific aptamercontrols were performed. FIG. 6A shows a clear concentration-dependentinhibition of polymerization by the deoxy-3A aptamer. Additionally, thedose response was quantified by determining the slope of thepolymerization curves as a function of deoxy 3-A aptamer concentration(FIG. 6B). The slopes, as measured from time points 22-50 minutes in thedose response curves, were calculated and plotted as a function ofaptamer concentration to display the dose-dependent effects of thisaptamer in inhibiting polymerization.

Lipofectin Facilitates Entry of Deoxy 3-A Aptamer into Sickle Red BloodCells

To determine if lipofectin could facilitate entry of the deoxy 3-Aaptamer into sickle red blood cells, sickle red blood cells obtainedwith consent from patients were washed and subjected to transfectionusing a standard protocol with Lipofectin reagent in the presence orabsence of fluorescently labeled deoxy 3-A aptamer. The cells werevigorously washed in high salt buffer following transfection to removeany non-specifically bound aptamer. Increased fluorescence intensity ofthe population demonstrates successful transfer of deoxy 3-A into redblood cells. Fluorescent microscopy following transfection confirmed theintroduction of the deoxy 3-A aptamer into a subset of the red bloodcells (FIG. 7).

HbS Retains the Ability to Form New Polymer when Growing Filament Endsare Provided by Mechanical Disruption

To evaluate the ability of HbS to form new polymer when growing filamentends were provided by mechanical disruption, polymerization assays wereperformed as previously described. At the end of the assay, cuvettescontaining HbS or HbS plus aptamer were shaken in an effort to breaklong filaments and supply new ends for a polymerization reaction. HbS inthe presence of the deoxy 3-A aptamer was capable of forming newpolymer, demonstrating that the aptamer is not merely causing proteindenaturation over time as a mechanism of action (FIG. 8).

REFERENCES

All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

1. A polynucleotide aptamer that specifically binds sickle hemoglobin(HbS).
 2. The polynucleotide aptamer of claim 1, wherein thepolynucleotide aptamer is an RNA aptamer.
 3. The polynucleotide aptamerof claim 2, wherein the polynucleotide aptamer inhibits polymerizationof HbS.
 4. The polynucleotide aptamer of claim 3, wherein thepolynucleotide aptamer specifically binds oxygenated HbS (oxy-HbS). 5.The polynucleotide aptamer claim 3, wherein the polynucleotide aptamerspecifically binds deoxygenated HbS (deoxy-HbS).
 6. The polynucleotideaptamer of claim 3, wherein the polynucleotide aptamer specificallybinds both oxygenated HbS (oxy-HbS) and deoxygenated HbS (deoxy-HbS). 7.The polynucleotide aptamer of claim 2, comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence atleast 80% identical to any one of SEQ ID NOS:2-60; (b) a nucleotidesequence at least 90% identical to any one of SEQ ID NOS:2-60; (c) anucleotide sequence at least 95% identical to any one of SEQ IDNOS:2-60; (d) a nucleotide sequence at least 99% identical to any one ofSEQ ID NOS:2-60; and (e) the nucleotide sequence of any one of SEQ IDNOS:2-60.
 8. The polynucleotide aptamer of claim 2, comprising anucleotide sequence selected from the group consisting of: (a) anucleotide sequence at least 80% identical to any one of SEQ ID NOS:2,4, 31, and 37; (b) a nucleotide sequence at least 90% identical to anyone of SEQ ID NOS:2, 4, 31, and 37; (c) a nucleotide sequence at least95% identical to any one of SEQ ID NOS:2, 4, 31, and 37; (d) anucleotide sequence at least 99% identical to any one of SEQ ID NOS:2,4, 31, and 37; and (e) the nucleotide sequence of any one of SEQ IDNOS:2, 4, 31, and
 37. 9. The polynucleotide aptamer of claim 7 selectedfrom the group consisting of: (a) wherein when the polynucleotideaptamer is any one of SEQ ID NOS:2, 4, or 5, the polynucleotide aptamerfurther comprises a consensus sequence consisting of SEQ ID NO:61; (b)wherein when the polynucleotide aptamer is any one of SEQ ID NOS:11 or14, the polynucleotide aptamer further comprises a consensus sequenceconsisting of SEQ ID NO:62; (c) wherein when the polynucleotide aptameris any one of SEQ ID NOS: 37, 38, 40, or 49, the polynucleotide aptamerfurther comprises a consensus sequence consisting of SEQ ID NO:63; (d)wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 31,37, 38, 40, 42, 45, 46, 47, 48, 49, 53, 56, 59 or 60, the polynucleotideaptamer further comprises a consensus sequence consisting of SEQ IDNO:64; and (e) wherein when the polynucleotide aptamer is any one of SEQID NOS: 2, 4, 5, 8, 34, or 57, the polynucleotide aptamer furthercomprises a consensus sequence consisting of SEQ ID NO:65.
 10. Thepolynucleotide aptamer of claim 2, comprising a consensus sequenceconsisting of a nucleotide sequence selected from the group consistingof SEQ ID NOS:61, 62, 63, 64, and
 65. 11. A polynucleotide aptamer ofclaim 1, wherein the polynucleotide aptamer is modified to increase itscirculating half-life after administration to a subject.
 12. Apolynucleotide encoding the polynucleotide aptamer of claim
 1. 13. Avector comprising the polynucleotide of claim
 12. 14. A cell comprisingthe polynucleotide aptamer of claim
 1. 15. A method of treating orpreventing sickle cell disease in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of a polynucleotide aptamer that specifically binds sicklehemoglobin (HbS), wherein the polynucleotide aptamer inhibitspolymerization of HbS.
 16. The method of claim 15, wherein thepolynucleotide aptamer is an RNA aptamer.
 17. The method of claim 16,wherein the polynucleotide aptamer specifically binds oxygenated HbS(oxy-HbS).
 18. The method of claim 16, wherein the polynucleotideaptamer specifically binds deoxygenated HbS (deoxy-HbS).
 19. The methodof claim 16, wherein the polynucleotide aptamer specifically binds bothoxygenated HbS (oxy-HbS) and deoxygenated HbS (deoxy-HbS).
 20. Themethod of claim 16, wherein the polynucleotide aptamer comprises anucleotide sequence selected from the group consisting of: (a) anucleotide sequence at least 80% identical to any one of SEQ IDNOS:2-60; (b) a nucleotide sequence at least 90% identical to any one ofSEQ ID NOS:2-60; (c) a nucleotide sequence at least 95% identical to anyone of SEQ ID NOS:2-60; (d) a nucleotide sequence at least 99% identicalto any one of SEQ ID NOS:2-60; and (e) the nucleotide sequence of anyone of SEQ ID NOS:2-60.
 21. The method of claim 16, wherein thepolynucleotide aptamer comprises a nucleotide sequence selected from thegroup consisting of: (a) a nucleotide sequence at least 80% identical toany one of SEQ ID NOS:2, 4, 31, and 37; (b) a nucleotide sequence atleast 90% identical to any one of SEQ ID NOS:2, 4, 31, and 37; (c) anucleotide sequence at least 95% identical to any one of SEQ ID NOS:2,4, 31, and 37; (d) a nucleotide sequence at least 99% identical to anyone of SEQ ID NOS:2, 4, 31, and 37; and (e) the nucleotide sequence ofany one of SEQ ID NOS:2, 4, 31, and
 37. 22. The method of either ofclaim 20, wherein the polynucleotide aptamer is selected from the groupconsisting of: (a) wherein when the polynucleotide aptamer is any one ofSEQ ID NOS:2, 4, or 5, the polynucleotide aptamer further comprises aconsensus sequence consisting of SEQ ID NO:61; (b) wherein when thepolynucleotide aptamer is any one of SEQ ID NOS:11 or 14, thepolynucleotide aptamer further comprises a consensus sequence consistingof SEQ ID NO:62; (c) wherein when the polynucleotide aptamer is any oneof SEQ ID NOS: 37, 38, 40, or 49, the polynucleotide aptamer furthercomprises a consensus sequence consisting of SEQ ID NO:63; (d) whereinwhen the polynucleotide aptamer is any one of SEQ ID NOS: 31, 37, 38,40, 42, 45, 46, 47, 48, 49, 53, 56, 59 or 60, the polynucleotide aptamerfurther comprises a consensus sequence consisting of SEQ ID NO:64; and(e) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 2,4, 5, 8, 34, or 57, the polynucleotide aptamer further comprises aconsensus sequence consisting of SEQ ID NO:65.
 23. The method of claim16, wherein the polynucleotide aptamer comprises a consensus sequenceconsisting of a nucleotide sequence selected from the group consistingof SEQ ID NOS:61, 62, 63, 64, and
 65. 24. The method of claim 16,wherein the polynucleotide aptamer is modified to increase itscirculating half-life after administration to the subject.
 25. Themethod of claim 16, wherein the polynucleotide aptamer is in apharmaceutically acceptable carrier.
 26. The method of claim 16, whereinthe sickle cell disease is sickle cell anemia.
 27. The method of claim16, further comprising contacting the polynucleotide aptamer with anantidote.
 28. The method of claim 27, wherein the antidote is anoligonucleotide comprising a sequence complementary to at least aportion of the polynucleotide aptamer.
 29. A method for diagnosing orpredicting a sickle cell disease in a subject having or at risk ofdeveloping a sickle cell disease or at risk of passing it on tooffspring, the method comprising: (a) obtaining a biological sample fromthe subject; (b) contacting the biological sample with a polynucleotideaptamer that specifically binds to HbS; and (c) detecting binding of thepolynucleotide aptamer with HbS in the biological sample; whereindetection of binding of the polynucleotide aptamer with HbS in thebiological sample is indicative of the subject having or at risk ofdeveloping a sickle cell disease or at risk of passing it on tooffspring.
 30. The method of claim 29, wherein the polynucleotideaptamer is an RNA aptamer.
 31. The method of claim 30, wherein thepolynucleotide aptamer specifically binds oxygenated HbS (oxy-HbS). 32.The method of claim 30, wherein the polynucleotide aptamer specificallybinds deoxygenated HbS (deoxy-HbS).
 33. The method of claim 30, whereinthe polynucleotide aptamer specifically binds both oxygenated HbS(oxy-HbS) and deoxygenated HbS (deoxy-HbS).
 34. The method of claim 30,wherein the polynucleotide aptamer comprises a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence atleast 80% identical to any one of SEQ ID NOS:2-60; (b) a nucleotidesequence at least 90% identical to any one of SEQ ID NOS:2-60; (c) anucleotide sequence at least 95% identical to any one of SEQ IDNOS:2-60; (d) a nucleotide sequence at least 99% identical to any one ofSEQ ID NOS:2-60; and (e) the nucleotide sequence of any one of SEQ IDNOS:2-60.
 35. The method of claim 30, wherein the polynucleotide aptamercomprises a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence at least 80% identical to any one of SEQ IDNOS:2, 4, 31, and 37; (b) a nucleotide sequence at least 90% identicalto any one of SEQ ID NOS:2, 4, 31, and 37; (c) a nucleotide sequence atleast 95% identical to any one of SEQ ID NOS:2, 4, 31, and 37; (d) anucleotide sequence at least 99% identical to any one of SEQ ID NOS:2,4, 31, and 37; and (e) the nucleotide sequence of any one of SEQ IDNOS:2, 4, 31, and
 37. 36. The method of claim 34, wherein thepolynucleotide aptamer is selected from the group consisting of: (a)wherein when the polynucleotide aptamer is any one of SEQ ID NOS:2, 4,or 5, the polynucleotide aptamer further comprises a consensus sequenceconsisting of SEQ ID NO:61; (b) wherein when the polynucleotide aptameris any one of SEQ ID NOS:11 or 14, the polynucleotide aptamer furthercomprises a consensus sequence consisting of SEQ ID NO:62; (c) whereinwhen the polynucleotide aptamer is any one of SEQ ID NOS: 37, 38, 40, or49, the polynucleotide aptamer further comprises a consensus sequenceconsisting of SEQ ID NO:63; (d) wherein when the polynucleotide aptameris any one of SEQ ID NOS: 31, 37, 38, 40, 42, 45, 46, 47, 48, 49, 53,56, 59 or 60, the polynucleotide aptamer further comprises a consensussequence consisting of SEQ ID NO:64; and (e) wherein when thepolynucleotide aptamer is any one of SEQ ID NOS: 2, 4, 5, 8, 34, or 57,the polynucleotide aptamer further comprises a consensus sequenceconsisting of SEQ ID NO:65.
 37. The method of claim 30, wherein thepolynucleotide aptamer comprises a consensus sequence consisting of anucleotide sequence selected from the group consisting of SEQ ID NOS:61,62, 63, 64, and
 65. 38. The method of claim 30, wherein the sickle celldisease is sickle cell anemia.
 39. The method of claim 30, wherein thebiological sample comprises whole blood, hemocytes, serum, or plasma.40. The methods of claim 30, wherein the polynucleotide aptamer islabeled for detection with a fluorescent, luminescent, phosphorescent,radioactive, or colorimetric compound.
 41. A method of purifyinghemoglobin from a biological sample, the method comprising: (a)providing a biological sample containing hemoglobin; (b) contacting thebiological sample with a polynucleotide aptamer that specifically bindsto HbS under conditions effective to bind hemoglobin to the aptamer; and(c) recovering the hemoglobin bound to the aptamer.
 42. The method ofclaim 41, wherein the step of contacting the biological sample with thepolynucleotide aptamer that specifically binds to HbS under conditionseffective to bind hemoglobin to the polynucleotide aptamer comprisesproviding a solid support comprising an aptamer that specifically bindsto HbS immobilized onto the solid support through a spacer.
 43. Themethod of claim 41, wherein the polynucleotide aptamer is an RNAaptamer.
 44. The method of claim 43, wherein the polynucleotide aptamerspecifically binds oxygenated HbS (oxy-HbS).
 45. The method of claim 43,wherein the polynucleotide aptamer specifically binds deoxygenated HbS(deoxy-HbS).
 46. The method of claim 43, wherein the polynucleotideaptamer specifically binds both oxygenated HbS (oxy-HbS) anddeoxygenated HbS (deoxy-HbS).
 47. The method of claim 43, wherein thepolynucleotide aptamer comprises a nucleotide sequence selected from thegroup consisting of: (a) a nucleotide sequence at least 80% identical toany one of SEQ ID NOS:2-60; (b) a nucleotide sequence at least 90%identical to any one of SEQ ID NOS:2-60; (c) a nucleotide sequence atleast 95% identical to any one of SEQ ID NOS:2-60; (d) a nucleotidesequence at least 99% identical to any one of SEQ ID NOS:2-60; and (e)the nucleotide sequence of any one of SEQ ID NOS:2-60.
 48. The method ofclaim 43, wherein the polynucleotide aptamer comprises a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence at least 80% identical to any one of SEQ ID NOS:2, 4, 31, and37; (b) a nucleotide sequence at least 90% identical to any one of SEQID NOS:2, 4, 31, and 37; (c) a nucleotide sequence at least 95%identical to any one of SEQ ID NOS:2, 4, 31, and 37; (d) a nucleotidesequence at least 99% identical to any one of SEQ ID NOS:2, 4, 31, and37; and (e) the nucleotide sequence of any one of SEQ ID NOS:2, 4, 31,and
 37. 49. The method of claim 47, wherein the polynucleotide aptameris selected from the group consisting of: (a) wherein when thepolynucleotide aptamer is any one of SEQ ID NOS:2, 4, or 5, thepolynucleotide aptamer further comprises a consensus sequence consistingof SEQ ID NO:61; (b) wherein when the polynucleotide aptamer is any oneof SEQ ID NOS:11 or 14, the polynucleotide aptamer further comprises aconsensus sequence consisting of SEQ ID NO:62; (c) wherein when thepolynucleotide aptamer is any one of SEQ ID NOS: 37, 38, 40, or 49, thepolynucleotide aptamer further comprises a consensus sequence consistingof SEQ ID NO:63; (d) wherein when the polynucleotide aptamer is any oneof SEQ ID NOS: 31, 37, 38, 40, 42, 45, 46, 47, 48, 49, 53, 56, 59 or 60,the polynucleotide aptamer further comprises a consensus sequenceconsisting of SEQ ID NO:64; and (e) wherein when the polynucleotideaptamer is any one of SEQ ID NOS: 2, 4, 5, 8, 34, or 57, thepolynucleotide aptamer further comprises a consensus sequence consistingof SEQ ID NO:65.
 50. The method of claim 43, wherein the polynucleotideaptamer comprises a consensus sequence consisting of a nucleotidesequence selected from the group consisting of SEQ ID NOS:61, 62, 63,64, and
 65. 51. The method of claim 43, wherein the polynucleotideaptamer is modified to enable covalent immobilization or to preventenzymatic degradation.
 52. The method of claim 43, wherein thebiological sample comprises whole blood, hemocytes, serum, or plasma.53. A method of using a three-dimensional structure of a polynucleotideaptamer that specifically binds to HbS and inhibits polymerization ofHbS in a drug screening assay comprising: (a) selecting a potential drugby performing rational drug design with the three-dimensional structureof the polynucleotide aptamer that specifically binds to HbS andinhibits polymerization of HbS determined from one or more sets ofatomic coordinates; wherein said selecting is performed in conjunctionwith computer modeling; (b) contacting the potential drug with HbS; (c)detecting the binding of the potential drug with the HbS; and (d)detecting the inhibition of polymerization of HbS by the potential drug;wherein a potential drug is selected as a drug if the potential drugbinds to HbS and inhibits polymerization of HbS.
 54. The method of claim53, wherein the polynucleotide aptamer is an RNA aptamer.
 55. The methodof claim 54, wherein the polynucleotide aptamer specifically bindsoxygenated HbS (oxy-HbS).
 56. The method of claim 54, wherein thepolynucleotide aptamer specifically binds deoxygenated HbS (deoxy-HbS).57. The method of claim 54, wherein the polynucleotide aptamerspecifically binds both oxygenated HbS (oxy-HbS) and deoxygenated HbS(deoxy-HbS).
 58. The method of claim 54, wherein the polynucleotideaptamer comprises a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence at least 80% identical to anyone of SEQ ID NOS:2-60; (b) a nucleotide sequence at least 90% identicalto any one of SEQ ID NOS:2-60; (c) a nucleotide sequence at least 95%identical to any one of SEQ ID NOS:2-60; (d) a nucleotide sequence atleast 99% identical to any one of SEQ ID NOS:2-60; and (e) thenucleotide sequence of any one of SEQ ID NOS:2-60.
 59. The method ofclaim 54, wherein the polynucleotide aptamer comprises a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence at least 80% identical to any one of SEQ ID NOS:2, 4, 31, and37; (b) a nucleotide sequence at least 90% identical to any one of SEQID NOS:2, 4, 31, and 37; (c) a nucleotide sequence at least 95%identical to any one of SEQ ID NOS:2, 4, 31, and 37; (d) a nucleotidesequence at least 99% identical to any one of SEQ ID NOS:2, 4, 31, and37; and (e) the nucleotide sequence of any one of SEQ ID NOS:2, 4, 31,and
 37. 60. The method of claim 58, wherein the polynucleotide aptameris selected from the group consisting of: (a) wherein when thepolynucleotide aptamer is any one of SEQ ID NOS:2, 4, or 5, thepolynucleotide aptamer further comprises a consensus sequence consistingof SEQ ID NO:61; (b) wherein when the polynucleotide aptamer is any oneof SEQ ID NOS:11 or 14, the polynucleotide aptamer further comprises aconsensus sequence consisting of SEQ ID NO:62; (c) wherein when thepolynucleotide aptamer is any one of SEQ ID NOS: 37, 38, 40, or 49, thepolynucleotide aptamer further comprises a consensus sequence consistingof SEQ ID NO:63; (d) wherein when the polynucleotide aptamer is any oneof SEQ ID NOS: 31, 37, 38, 40, 42, 45, 46, 47, 48, 49, 53, 56, 59 or 60,the polynucleotide aptamer further comprises a consensus sequenceconsisting of SEQ ID NO:64; and (e) wherein when the polynucleotideaptamer is any one of SEQ ID NOS: 2, 4, 5, 8, 34, or 57, thepolynucleotide aptamer further comprises a consensus sequence consistingof SEQ ID NO:65.
 61. The method of claim 54, wherein the polynucleotideaptamer comprises a consensus sequence consisting of a nucleotidesequence selected from the group consisting of SEQ ID NOS: 61, 62, 63,64, and 65.